Modified ligand-gated ion channels and methods of use

ABSTRACT

This document relates to materials and methods for controlling ligand gated ion channel (LGIC) activity. For example, modified LGICs including at least one LGIC subunit having a modified ligand binding domain (LBD) and/or a modified ion pore domain (IPD) are provided. Also provided are exogenous LGIC ligands that can bind to and activate the modified LGIC, as well as methods of modulating ion transport across the membrane of a cell of a mammal, methods of modulating the excitability of a cell in a mammal, and methods of treating a mammal having a channelopathy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.16/868,205, filed on May 6, 2020, which is a Continuation of U.S. patentapplication Ser. No. 15/644,295, filed on Jul. 7, 2017, which claims thebenefit of U.S. Patent Application Ser. No. 62/486,779, filed on Apr.18, 2017, and claims the benefit of U.S. Patent Application Ser. No.62/359,534, filed on Jul. 7, 2016. The disclosures of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an ASCII text file named 30872-0014005_ST25.txt. TheASCII text file, created on Aug. 10, 2021, is 34 kilobytes in size. Thematerial in the ASCII text file is hereby incorporated by reference inits entirety.

BACKGROUND 1. Technical Field

This document relates to materials and methods for controlling ligandgated ion channel (LGIC) activity. For example, this document providesmodified LGICs including at least one LGIC subunit having a modifiedligand binding domain (LBD) and/or a modified ion pore domain (IPD).Also provided are exogenous LGIC ligands that can bind to and activatethe modified LGIC. In some cases, a modified LGIC and an exogenousligand can be used to treat a mammal having a channelopathy (e.g., aneural channelopathy or a muscle channelopathy). In some cases, amodified LGIC and an exogenous LGIC ligand can be used to modulate(e.g., activate or inhibit) ion transport across the membrane of a cellof a mammal. In some cases, a modified LGIC and an exogenous LGIC ligandcan be used to modulate (e.g., increase or decrease) the excitability ofa cell in a mammal.

2. Background Information

Ion channels mediate ionic flux in cells, which profoundly affects theirbiological function. A prominent instance of this is in neurons, whereion channels control electrical signaling within between neurons toinfluence physiology, sensation, behavior, mood, and cognition.

Different LGICs have distinct ligand binding properties as well asspecific ion conductance properties (Hille 2001 Ion Channels ofExcitable Membranes. pp. 814. Sunderland, Mass.: Sinauer Associates;Kandel et al 2000 Principles of Neural Science. USA: McGraw-Hill Co.1414 pp). For example, nicotinic acetylcholine receptors (nAChRs) bindthe endogenous ligand acetylcholine (ACh), which activates conductancesfor cations and typically depolarizes cells, thereby increasing cellularexcitability. In contrast, the glycine receptor (GlyR) binds theendogenous ligand glycine, which activates chloride anion conductanceand typically reduces the excitability of cells by hyperpolarizationand/or by an electrical shunt of the cellular membrane resistance.

SUMMARY

Levels of endogenous LGIC agonists such as ACh are not readilycontrolled.

This document provides materials and methods for controlling LGICactivity (e.g., increasing the sensitivity of LGICs to exogenous ligandsand/or reducing sensitivity to endogenous ligands such as ACh). Forexample, this document provides modified LGICs including at least onemodified LGIC subunit having a LBD and an IPD, and having at least onemodified amino acid (e.g., an amino acid substitution). Also providedare exogenous LGIC ligands that can bind to and activate the modifiedLGIC. In some cases, a modified LGIC and an exogenous ligand can be usedto treat a mammal having a channelopathy (e.g., a neural channelopathyor a muscle channelopathy). In some cases, a modified LGIC and anexogenous LGIC ligand can be used to modulate (e.g., activate orinhibit) ion transport across the membrane of a cell of a mammal. Insome cases, a modified LGIC and an exogenous LGIC ligand can be used tomodulate (e.g., increase or decrease) the excitability of a cell in amammal.

Having the ability to control LGIC activity provides a unique andunrealized opportunity to achieve control of ion transport in cells. Forexample, modified LGICs having increased sensitivity for one or moreexogenous LGIC ligands can be used to provide temporal and spatialcontrol of ion transport and/or cellular excitability based on deliveryof the exogenous LGIC ligand. For example, modified LGICs with reducedsensitivity for endogenous LGIC ligands prevent unwanted activation ofmodified LGICs and allow for selective control over the modified LGIC byexogenous ligands. Further, exogenous LGIC ligands having increasedpotency for a modified LGIC improve selectivity of targeting of themodified LGIC over endogenous ion channels. Thus, the modified LCIGs andexogenous LGIC ligands provided herein are useful to achieve atherapeutic effect while reducing side effects from the small moleculeson unintended targets.

As described herein, one or more mutations in a modified LGIC canenhance potency for exogenous LGIC ligands. Mutation of the α7 LBD ofα7-GlyR at residue L131 (e.g., substituting Leu with Gly or Ala)increased potency for varenicline (16-fold) and tropisetron (3.6-fold)while reducing ACh potency (−6.4-fold) relative to α7-GlyR. Mutation ofα7 LBD of α7-GlyR at residue G175 (e.g., G175K) or P216 (e.g., P216I)enhanced potency for ACh, nicotine, tropisetron, varenicline, as well asother quinuclidine and tropane agonists. Combining the mutation atresidue G175K with mutations that reduce potency of the endogenousagonist ACh (e.g. Y115F) produced α7-GlyR Y115F G175K with increasedpotency for tropisetron (5.5-fold) and reduced potency from ACh(−8-fold). In addition, combining mutations in the α7 LBD at residues 77(e.g., substituting Trp with Phe or Tyr) and/or 79 (e.g., substitutingGln with Gly, Ala, or Ser) and/or 131 (e.g., substituting Leu with Glyor Ala) and/or 141 (e.g., substituting Leu with Phe or Pro) in thesechimeric channels with potency enhancing mutations at residues G175(e.g., G175K) or P216 (e.g., P216I) increase potency for distinctligands and/or reduce ACh potency. For example, a chimeric α7-GlyR LGICwith a α7 nAChR LBD (α7 LBD) having a mutation at residue 79 (e.g.,substituting Gln with Gly), a mutation at residue 115 (e.g.,substituting Tyr with Phe), and a mutation at residue 175 (e.g.,substituting Gly with Lys) has greater than 100-fold increasedsensitivity to an exogenous tropane LGIC ligand compound 723 (atropane), and reduced ACh sensitivity (−15-fold) relative to theunmodified chimeric α7-GlyR LGIC. Furthermore, a modified LGIC includingat least one chimeric LGIC subunit having an α7 nAChR LBD (α7 LBD)having a mutation at residue 79 (e.g., substituting Gln with Ala, Gly,or Ser) and a GlyR IPD having a mutation at residue 298 (e.g.,substituting Ala with Gly) has nearly 20-fold increased sensitivity foran exogenous LGIC ligand, such as a quinuclidine or a tropane.Additional mutations at residue 27 (e.g., substituting Arg with Asp) and41 (e.g., substituting Glu with Arg) of the α7 LBD reduced theassociation of the modified chimeric LGIC with an unmodified ionchannels. Additional mutations at residue 115 (e.g., substituting Tyrwith Phe), 139 (e.g., substituting Gln with Gly or Leu), 210 (e.g.,substituting Tyr with Phe) 217 (e.g., substituting Tyr with Phe), and/or219 (e.g., substituting Asp with Ala) of the α7 LBD reduced sensitivityof the chimeric LGIC to the endogenous ligand ACh. These chimeric LGICsallow for highly selective control over cellular function in cells of amammal while minimizing cross-reactivity with endogenous signalingsystems in the mammal.

In general, one aspect of this document features a modified LGIC havingat least one modified LGIC subunit which includes a LBD having an aminoacid modification, and an IPD, where an exogenous LGIC ligand activatesthe modified LGIC. The modified LGIC can be a chimeric LGIC having a LBDfrom a first LGIC and an IPD from a second LGIC. The LBD can be analpha7 nicotinic acetylcholine receptor (α7-nAChR) LBD. The modifiedLGIC of claim 3, wherein the at least one modified amino acid in theα7-nAChR LBD comprises an amino acid substitution at an amino acidresidue selected from the group consisting of residues 77, 79, 131, 139,141, 175, and 216 of the α7-nAChR LBD. The amino acid substitution canbe at residue 79 of the α7 LBD, and the amino acid substitution can beQ79A, Q79Q or Q79S. For example, the amino acid substitution at residue79 of the α7 LBD can be Q79G The IPD can be a serotonin 3 receptor(5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid(GABA) receptor IPD, or an α7-nAChR IPD. The IPD can be a GlyR IPD, andthe GlyR IPD can include an amino acid substitution at residue 298(e.g., a A298G substitution) of the chimeric LGIC. The IPD can be a GABAIPD, and the GABA IPD can include an amino acid substitution at residue298 (e.g., a W298A substitution) of the modified LGIC. The modified LGICcan be a chimeric LGIC including an α7 LBD having a Q79G amino acidsubstitution, and a GlyR IPD having a A298G amino acid substitution. Theexogenous LGIC ligand can be a synthetic exogenous LGIC ligand selectedfrom the group consisting of a quinuclidine, a tropane, a9-azabicyclo[3.3.1]nonane, a6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and a1,4-diazabicyclo[3.2.2]nonane. When the synthetic exogenous LGIC ligandis a tropane, the tropane can be tropisetron, pseudo-tropisetron,nortropisetron, compound 723, compound 725, compound 737, or compound745. When the synthetic exogenous LGIC ligand is a quinuclidine, thequinuclidine can be PNU-282987, PHA-543613, compound 0456, compound0434, compound 0436, compound 0354, compound 0353, compound 0295,compound 0296, compound 0536, compound 0676, or compound 702. When thesynthetic exogenous LGIC ligand is a6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, theligand can be compound 765 or compound 770. When the synthetic exogenousLGIC ligand is a 1,4-diazabicyclo[3.2.2]nonane, the ligand can becompound 773 or compound 774. In some cases, the LBD can be an α7 LBD,and the α7 LBD can also include at least one modified amino acid thatconfers selective binding to another α7 LBD having the at least onemodified amino acid over binding to an unmodified LGIC. The unmodifiedLGIC can be an endogenous LGIC (e.g., an endogenous α7-nAChR). The atleast one modified amino acid in the α7 LBD that confers reduced bindingto the unmodified LGIC can include an amino acid substitution at residue27 (e.g., a R27D substitution) and/or residue 41 (e.g., an E41Rsubstitution). In some cases, the IPD can be a 5HT3 IPD, and the 5HT3IPD can include at least one modified amino acid that confers increasedion conductance to the modified LGIC. The at least one modified aminoacid in the 5HT3 IPD that confers increased ion conductance to themodified LGIC can include an amino acid substitution at an amino acidresidue at residue 425 (e.g., a R425Q substitution), 429 (e.g., a R429Dsubstitution), and/or 433 (e.g., a R433A substitution).

In another aspect, this document features a modified LGIC having atleast one modified LGIC subunit including a LBD having at least onemodified amino acid, and an IPD, where the at least one modified aminoacid in the LBD reduces binding with an endogenous LGIC ligand. Themodified LGIC can be a chimeric LGIC having a LBD from a first LGIC andan IPD from a second LGIC. The endogenous LGIC ligand can be ACh. Themodified LGIC can have an EC50 of greater than 20 μM for Ach. The atleast one modified amino acid can include an amino acid substitution atresidue 115, 139, 210, 217, and/or 219. When the at least one modifiedamino acid includes an amino acid substitution at residue 115, the aminoacid substitution can be a Y115F substitution. When the at least onemodified amino acid includes an amino acid substitution at residue 139,the amino acid substitution can be a Q139G or a Q139L substitution. Whenthe at least one modified amino acid includes an amino acid substitutionat residue 210, the amino acid substitution can be a Y210F substitution.When the at least one modified amino acid includes an amino acidsubstitution at residue 217, the amino acid substitution can be a Y217Fsubstitution. When the at least one modified amino acid includes anamino acid substitution at residue 219, the amino acid substitution canbe a D219A substitution.

In another aspect, this document features a ligand having increasedpotency for a modified ligand gated ion channel (LGIC), wherein theligand comprises Formula I:

where each of X1, X2, and X3 can independently be CH, CH2, O, NH, orNMe; where each n can independently be 0 or 1; where Y=O or S; whereA=an aromatic substituent; and where R=H or pyridinylmethylene. Thearomatic substituent can be 1H-indole, 4-(trifluoromethyl) benzene,2,5-dimethoxy benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl) nicotinic, or 4-chloro-benzene.

In some cases, a LGIC ligand can be a quinuclidine and can have astructure shown in Formula II:

where X3=O, NH, or CH2; where Y=O or S; where A=an aromatic substituent;and where R=H or pyridinylmethylene. The aromatic substituent can be1H-indole, 4-(trifluoromethyl) benzene, 4-chloro benzene, 2,5-dimethoxybenzene, 4-(trifluoromethyl) benzene, 4-chloroaniline, aniline,5-(trifluoromethyl) pyridin-2-yl, 6-(trifluoromethyl) nicotinic,3-chloro-4-fluoro benzene, or 1H-indole. The quinuclidine can bePNU-282987, PHA-543613, compound 0456, compound 0434, compound 0436,compound 0354, compound 0353, compound 0295, compound 0296, compound0536, compound 0676, or compound 702.

In some cases, a LGIC ligand can be a tropane and can have a structureshown in Formula III:

where X2=NH or NMe; where X3=O, NH, or CH2; where Y=O or S; and whereA=an aromatic substituent. The aromatic substituent can be 1H-indole,7-methoxy-1H-indole, 7-methyl-1H-indole, 5-chloro-1H-indole, or1H-indazole. The tropane can be tropisetron, pseudo-tropisetron,nortropisetron, compound 723, compound 725, compound 737, or compound745.

In some cases, a LGIC ligand can be a 9-azabicyclo[3.3.1]nonane and canhave a structure shown in Formula IV:

where X1 can be CH, X2 can be NH or NMe, X3 can be O, NH, or CH; Y canbe O or S, and A can be an aromatic substituent. The aromaticsubstituent can be 4-chloro-benzene. The 9-azabicyclo[3.3.1]nonane canbe compound 0536.

In another aspect, this document features a ligand having increasedpotency for a modified ligand gated ion channel (LGIC), where the ligandcan be a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepineand have a structure shown in Formula V:

where R can be H or CH3, and where A can be H or an aromaticsubstituent. The6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine can bevarenicline, compound 0765, or compound 0770.

In another aspect, this document features a ligand having increasedpotency for a modified ligand gated ion channel (LGIC), where the ligandcan be a 1,4-diazabicyclo[3.2.2]nonane and can have a structure shown inFormula VI:

where R can be H, F, or NO₂. The 1,4-diazabicyclo[3.2.2]nonane can be3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide,compound 0773, or compound 0774.

In another aspect, this document features methods of treating achannelopathy in a mammal. The methods include, or consist essentiallyof, administering to a cell in the mammal a modified LGIC, where anexogenous LGIC ligand selectively binds the modified LGIC. The modifiedLGIC has at least one modified LGIC subunit including a LBD including atleast one modified amino acid, and an IPD; and administering theexogenous ligand to the mammal. The channelopathy can be Barttersyndrome, Brugada syndrome, catecholaminergic polymorphic ventriculartachycardia (CPVT), congenital hyperinsulinism, cystic fibrosis, Dravetsyndrome, episodic ataxia, erythromelalgia, generalized epilepsy (e.g.,with febrile seizures), familial hemiplegic migraine, fibromyalgia,hyperkalemic periodic paralysis, hypokalemic periodic paralysis,Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g., Romano-Wardsyndrome), short QT syndrome, malignant hyperthermia, mucolipidosis typeIV, myasthenia gravis, myotonia congenital, neuromyelitis optica,neuromyotonia, nonsyndromic deafness, paramyotonia congenital, retinitispigmentosa, timothy syndrome, tinnitus, seizure, trigeminal neuralgia,or multiple sclerosis.

In another aspect, this document features methods of modulating iontransport across a cell membrane of a mammal. The methods include, orconsist essentially of, administering to the cell a modified LGIC, wherean exogenous LGIC ligand selectively binds the modified LGIC. Themodified LGIC has at least one modified LGIC subunit including a LBDincluding at least one modified amino acid, and an IPD; andadministering the exogenous ligand to the mammal. The modulating caninclude activating or inhibiting ion transport. The cell can be aneuron, a glial cell, a myocyte, a stem cell, an endocrine cell, or animmune cell. The administering the modified LGIC to the cell can be anin vivo administration or an ex vivo administration. The administeringthe modified LGIC to the cell can include administering a nucleic acidencoding the modified LGIC.

In another aspect, this document features methods of modulating theexcitability of a cell in a mammal. The methods include, or consistessentially of, administering to the cell from the mammal a modifiedLGIC, where an exogenous LGIC ligand selectively binds the modifiedLGIC. The modified LGIC has at least one modified LGIC subunit includinga LBD including at least one modified amino acid, and an IPD; andadministering the exogenous ligand to the mammal. The modulating caninclude increasing the excitability of the cell or decreasing theexcitability of the cell. The cell can be an excitable cell. The cellcan be a neuron, a glial cell, a myocyte, a stem cell, an endocrinecell, or an immune cell. The administering the modified LGIC to the cellcan be an in vivo administration or an ex vivo administration. Theadministering the modified LGIC to the cell can include administering anucleic acid encoding the modified LGIC.

In another aspect, this document features methods of modulating theactivity of a cell in a mammal. The methods include, or consistessentially of, administering to the cell a modified LGIC, where anexogenous LGIC ligand selectively binds the modified LGIC. The modifiedLGIC has at least one modified LGIC subunit including a LBD including atleast one modified amino acid, and an IPD; and administering theexogenous ligand to the mammal. The modulating can include increasingthe activity of the cell or decreasing the activity of the cell. Theactivity can be ion transport, passive transport, excitation,inhibition, or exocytosis. The cell can be a neuron, a glial cell, amyocyte, a stem cell, an endocrine cell, or an immune cell. Theadministering the modified LGIC to the cell can be an in vivoadministration or an ex vivo administration. The administering themodified LGIC to the cell can include administering a nucleic acid(e.g., via a viral vector such as an adeno-associated virus, a herpessimplex virus, or a lentivirus) encoding the modified LGIC.

In another aspect, this document features a method for identifying aligand that selectively binds to a modified LGIC. The method includes,or consists essentially of, providing one or more candidate ligands tothe modified LGIC described herein, and detecting binding between thecandidate ligand and the modified LGIC, thereby identifying a ligandthat selectively binds the modified LGIC. The modified LGIC can be ahomomeric modified LGIC.

In another aspect, this document features a method for detecting amodified LGIC. The method includes, or consists essentially of,providing one or more modified LGIC subunits described herein, providingan agent that selectively binds the modified LGIC, and detecting bindingbetween the modified LGIC and the agent that selectively binds themodified LGIC, thereby detecting the modified LGIC. The agent thatselectively binds the modified LGIC comprises can be antibody, a protein(e.g., bungarotoxin), or a small molecule (e.g., a positron emissiontomography (PET) ligand). The agent that selectively binds the modifiedLGIC can include a detectable label (e.g., a fluorescent label, aradioactive label, or a positron emitting label).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show exemplary amino acid sequences of chimeric LGICs.Mutation of amino acid residue 77 (e.g., W77F or W77Y) resulted insensitivity to granisetron and tropisetron. Mutation of amino acidresidue 79 (e.g., Q79G) was most effective for several agonists.Mutations of amino acid residue 131 (e.g., L131G, L131A, L131M, orL131N) altered sensitivity to varenicline, tropisetron, granisetron, andACh. Potency was considerably enhanced when LBD mutations were combinedwith mutation at amino acid residue 298 in the GlyR or GABAC IPD.Potency was also enhanced when α7 nAChR LBD mutations were combined withmutation at amino acid residue G175 and P216. FIG. 1A) An amino acidsequence of α7-5HT3 chimeric receptor (SEQ ID NO:6) including a human α7nAChR LBD (SEQ ID NO:1) and a murine 5HT3 IPD (SEQ ID NO:3) components.FIG. 1B) An amino acid sequence of α7-GlyR chimeric receptor (SEQ IDNO:7), including a human α7 nAChR LBD (SEQ ID NO:2) and a human GlyR IPD(SEQ ID NO:5) components. FIG. 1C) An amino acid sequence of α7-5HT3chimeric receptor (SEQ ID NO:8) including human α7 nAChR LBD (SEQ IDNO:1) and a human 5HT3 IPD (SEQ ID NO:4) components. FIG. 1D) An aminoacid sequence of α7-GABA_(C) chimeric receptor (SEQ ID NO:10) includinga human α7 nAChR LBD (SEQ ID NO:2) and a human GABA_(C) IPD (SEQ IDNO:9) components. FIG. 1E) An amino acid sequence of rat nAChR sequence(SEQ ID NO:12).

FIG. 2 shows EC50s for tropisetron against a α7-5HT3 chimeric LGIC andvariants of the chimeric LGIC with LBD mutations at positions noted inFIG. 1. Multiple mutations at Gln79 showed similar or improved potencyrelative to the unmodified α7-5HT3 channel (arrows).

FIGS. 3A-3B show the relative potency of known nAChR agonists forα7-5HT3 chimeric LGICs. FIG. 3A) A graph of EC50s normalized to theunmodified α7-5HT3 chimeric channel (log scale). *P<0.05, statisticallysignificant potency changes are noted (ANOVA followed by Dunn's test).FIG. 3B) Chemical structures of known nAChR agonists.

FIGS. 4A-4C show the relative potency of known nAChR agonists forα7-GlyR chimeric LGICs. FIG. 4A) A graph of EC50s for Q79 LBD mutantsnormalized to the unmodified α7-GlyR chimeric channel (log scale). FIG.4B) A graph of EC50s for A298G IPD mutation normalized to the unmodifiedα7-GlyR chimeric channel (log scale). FIG. 4C) A graph of EC50s forα7-GlyR^(A298G) normalized to the unmodified α7-GlyR chimeric channeland compared to the double mutant channel α7Q79G-GlyR^(A298G) (logscale). *P<0.05, statistically significant potency changes are noted(ANOVA followed by Dunn's test).

FIGS. 5A-5C show schematic structures of LGIC agonists with substitutionpatterns most compatible with potency enhancement for α7^(Q79G)-5HT3 andα7^(Q79G)-GlyR^(A298G). FIG. 5A) A generalized structure showingattributes associated with enhanced potency. FIG. 5B) Specificpharmacophores represented in (FIG. 5A) are quinuclidine, tropane, and9-azabicyclo[3.3.1]nonane core structures. FIG. 5C) Exemplary syntheticmolecules that show high potency for α7^(Q79G)-GlyR^(A298G),α7^(Q79G,Y115F,G175K)-GlyR, α7^(W77F,Q79G,G175K)-GlyR.

FIGS. 6A-6C show mutations that reduce association of chimeric LCIG α7nAChR LBDs with unmodified LBDs. FIG. 6A) Charge reversal schematicpotential configurations of transfecting two epitope tagged (HA and V5)constructs encoding α7-5HT3 (top) or two constructs encoding α7-5HT3-HAand α7^(R27D,E41R)-5HT3-V5 where association between the two differentepitope tagged subunits would be unfavored due to charge reversalmutations at the subunit interfaces. FIG. 6B) Whole cell recordings inHEK cells expressing α7^(R27D,E41R)-5HT3 with a V5 epitope tag showspotent responses to PNU-282987. FIG. 6C) Association of α7-5HT3 LGICswith HA and V5 epitope tags in HEK cells was probed by HAimmunoprecipitation (left) or total lysate isolation followed by westernblotting with either anti-HA (top) or anti-V5 antibodies (bottom). Incells co-expressing channels with the HA and V5 epitopes, anti-HA IPfollowed by anti-V5 immunoblotting shows the co-immunoprecipation ofunmodified channels of each type, but charge reversal mutations in theLBD α7^(R27D,E41R)-5HT3-V5 was not immunoprecipitated. MW of α7-5HT3 is˜48 kD (arrow).

FIG. 7 shows that chimeric LGICs can be controlled using an exogenousligand. Cortical neurons from a mouse brain transduced withα7^(Q79G)-GlyR^(A298G) chimeric LGIC via adeno-associated virus (AAV)vectors fires action potentials in response to 40 pA current injection(PRE) that are potently suppressed by 30 nM tropisetron. After washout(WASH) of tropisetron, neuron firing is restored.

FIGS. 8A-8C show activity of agonists on chimeric LGICs with a G175Kmutation. FIG. 8A) A graph of EC50s for Q79G G175K LBD mutants againstknown agonists normalized to the unmodified α7-GlyR chimeric channel(log scale). FIG. 8B) A graph of EC50s for ACh and tropisetron forchannels with mutations in α7-GlyR chimeric LGICs. Mutations that resultin channels with high potency for tropisetron and low potency for theendogenous ligand, acetylcholine (ACh) are optimal (grey shading).Unmod.: unmodified α7-GlyR chimeric LGIC. FIG. 8C) Action potentials ofcortical neurons from a mouse brain transduced withα7^(Q79G,Y115F,G175K)-GlyR chimeric LGIC. Neurons fire in response tocurrent injection (PRE) and are potently suppressed by 100 nMtropisetron. After washout (WASH) of tropisetron, neuron firing isrestored.

FIGS. 9A-9D show activity of agonists on chimeric LGICs with a L131Gmutation. FIG. 9A) A graph of EC50s for L131 LBD mutants against knownagonists normalized to the unmodified α7-GlyR chimeric channel (logscale). FIG. 9B) A graph of EC50s for ACh and tropisetron for channelswith mutations in α7^(L131G)-GlyR chimeric LGICs. FIG. 9C) A graphsshowing mutations that result in channels with high potency forvarenicline and low potency for the endogenous ligand, acetylcholine(ACh) are optimal (grey shading). Unmod.: unmodified α7-GlyR chimericLGIC. FIG. 9D) Action potentials of a cortical neuron from a mouse braintransduced with α7^(L131G,Q139L,Y217F)-GlyR chimeric LGIC. Neuron firesin response to current injection (PRE) and are potently suppressed by 10nM varenicline, even with >6-fold greater injected current. Afterwashout (WASH) of tropisetron, neuron firing is restored.

FIGS. 10A-10B show chemical structures of LGIC agonists. FIG. 10A)Chemical structures of LGIC agonists with substitution patterns mostcompatible with potency enhancement for α7^(Q79G,Yl15F,G175K)-GlyR. FIG.10B) Chemical structures of LGIC agonists with substitution patternsmost compatible with potency enhancement for α7^(L131G,Q139L,Y217F)-GlyRor α7^(L131G,Q139L,Y217F)-5HT3 HC.

DETAILED DESCRIPTION

This document provides modified LGICs and methods of using them. Forexample, this document provides modified LGICs including at least onemodified LGIC subunit having a LBD and an IPD, and having at least onemodified amino acid (e.g., an amino acid substitution). In some cases, amodified LGIC can be a chimeric LGIC. For example, a chimeric LGIC caninclude a LBD from a first LGIC and an IPD from a second LGIC. In somecases, the modified amino acid can confer pharmacological selectivity tothe modified LGIC. For example, the modified amino acid can confer themodified LGIC with selective binding of an exogenous LGIC ligand. Forexample, the modified amino acid can confer the modified LGIC withreduced (minimized or eliminated) binding of an unmodified LGIC subunit(an LGIC subunit lacking the modification and/or an endogenous LGICsubunit). For example, the modified amino acid can confer the modifiedLGIC with reduced (minimized or eliminated) binding of an endogenousLGIC ligand.

Modified LGICs provided herein can be used, for example, in methods fortreating channelopathies (e.g., a neural channelopathy or a musclechannelopathy). For example, a modified LGIC, and an exogenous LGICligand that can bind to and activate the modified LGIC, can be used totreat a mammal having a channelopathy. In some cases, a modified LGICand an exogenous LGIC ligand can be used to modulate (e.g., activate orinhibit) ion transport across the membrane of a cell of a mammal. Insome cases, a modified LGIC and an exogenous LGIC ligand can be used tomodulate (e.g., increase or decrease) the excitability of a cell in amammal.

Modified LGICs

As used herein a “modified” LGIC is an LGIC that includes at least oneLGIC subunit. A modified LGIC subunit can include at least one modifiedamino acid (e.g., an amino acid substitution) in the LBD and/or at leastone modified amino acid (e.g., an amino acid substitution) in the IPD. Amodified LGIC subunit described herein can be a modification of an LGICfrom any appropriate species (e.g., human, rat, mouse, dog, cat, horse,cow, goat, pig, or monkey). In some cases, a modified LGIC can includeat least one chimeric LGIC subunit having a non-naturally occurringcombination of a LBD from a first LGIC and an IPD from a second LGIC.

A modified LGIC can be a homomeric (e.g., having any number of the samemodified LGIC subunits) or heteromeric (e.g., having at least onemodified LGIC subunit and any number of different LGIC subunits). Insome cases, a modified LGIC described herein can be a homomeric modifiedLGIC. A modified LGIC described herein can include any suitable numberof modified LGIC subunits. In some cases, a modified LGIC can be atrimer, a tetramer, a pentamer, or a hexamer. For example, a modifiedLGIC described herein can be a pentamer.

A modified LGIC subunit described herein can be a modification of anyappropriate LGIC. The LGIC can conduct anions, cations, or both througha cellular membrane in response to the binding of a ligand. For example,the LGIC can transport sodium (Na+), potassium (K+), calcium (Ca2+),and/or chloride (Cl−) ions through a cellular membrane in response tothe binding of a ligand. Examples of LGICs include, without limitation,Cys-loop receptors (e.g., AChR such as a nAChR (e.g., a muscle-typenAChR or a neuronal-type nAChR), gamma-aminobutyric acid (GABA; such asGABA_(A) and GABA_(A)-ρ (also referred to as GABA_(C)) receptors, GlyR,GluC1 receptors, and 5HT3 receptors), ionotropic glutamate receptors(iGluR; such as AMPA receptors, kainate receptors, NMDA receptors, anddelta receptors), ATP-gated channels (e.g., P2X), andphosphatidylinositol 4,5-bisphosphate (PIP2)-gated channels. In caseswhere a modified LGIC described herein is a chimeric LGIC, the chimericLGIC can include a LBD selected from any appropriate LGIC and an IPDselected from any appropriate LGIC. In cases where a LGIC includesmultiple different subunits (for example, a neuronal-type nAChR includesα4, β2, and α7 subunits), the LBD and/or IPD can be selected from any ofthe subunits. For example, a LBD from a nAChR can be a α7 LBD. Arepresentative rat α7 nAChR amino acid sequence (including both a LBDand an IPD) is as follows.

SEQ ID NO: 12 MGGGRGGIWLALAAALLHVSLQGEFQRRLYKELVKNYNPLERPVANDSQPLTVYFSLSLLQIMDVDEKNQVLTTNIWLQMSWTDHYLQWNMSEYPGVKNVRFPDGQIWKPDILLYNSADERFDATFHTNVLVNASGHCQYLPPGIFKSSCYIDVRWFPFDVQQCKLKFGSWSYGGWSLDLQMQEADISSYIPNGEWDLMGIPGKRNEKFYECCKEPYPDVTYTVTMRRRTLYYGLNLLIPCVLISALALLVFLLPADSGEKISLGITVLLSLTVFMLLVAEIMPATSDSVPLIAQYFASTMIIVGLSVVVTVIVLRYHHHDPDGGKMPKWTRIILLNWCAWFLRMKRPGEDKVRPACQHKPRRCSLASVELSAGAGPPTSNGNLLYIGFRGLEGMHCAPTPDSGVVCGRLACSPTHDEHLMHGAHPSDGDPDLAKILEEVRYIANRNRCQDESEVICSEWKFAACVVDPLCLMAFSVFTIICTIGILMSAPNFVEAVSKDFA

In some cases, a modified LGIC subunit described herein can include aLBD from a α7 nAChR. Examples of α7 nAChR LBDs include, withoutlimitation, a human α7 nAChR LBD having the amino acid sequence setforth in SEQ ID NO:1, a human α7 nAChR LBD having the amino acidsequence set forth in SEQ ID NO:2, and a human α7 nAChR LBD having theamino acid sequence set forth in SEQ ID NO:11. In some cases, a α7 nAChRLBD can be a homolog, orthologue, or paralog of the human α7 nAChR LBDset forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:11. In some cases, aα7 nAChR LBD can be have at least 75 percent sequence identity (e.g., atleast 80%, at least 82%, at least 85%, at least 88%, at least 90%, atleast 93%, at least 95%, at least 97% or at least 99% sequence identity)to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:11.

SEQ ID NO: 1 MRCSPGGVWLALAASLLHVSLQGEFQRKLYKELVKNYNPLERPVANDSQPLTVYFSLSLLQIMDVDEKNQVLTTNIWLQMSWTDHYLQWNVSEYPGVKTVRFPDGQIWKPDILLYNSADERFDATFHTNVLVNSSGHCQYLPPGIFKSSCYIDVRWFPFDVQHCKLKFGSWSYGGWSLDLQMQEADISGYIPNGEWDLVGIPGK RSERFYECCKEPYPDVTFTVSEQ ID NO: 2 MRCSPGGVWLALAASLLHVSLQGEFQRKLYKELVKNYNPLERPVANDSQPLTVYFSLSLLQIMDVDEKNQVLTTNIWLQMSWTDHYLQWNVSEYPGVKTVRFPDGQIWKPDILLYNSADERFDATFHTNVLVNSSGHCQYLPPGIFKSSCYIDVRWFPFDVQHCKLKFGSWSYGGWSLDLQMQEADISGYIPNGEWDLVGIPGKRSERFYECCKEPYPDVTFTVTMRRR SEQ ID NO: 11MRCSPGGVWLALAASLLHVSLQGEFQRKLYKELVKNYNPLERPVANDSQPLTVYFSLSLLQIMDVDEKNQVLTTNIWLQMSWTDHYLQWNVSEYPGVKTVRFPDGQIWKPDILLYNSADERFDATFHTNVLVNSSGHCQYLPPGIFKSSCYIDVRWFPFDVQHCKLKFGSWSYGGWSLDLQMQEADISGYIPNGEWDLVGIPGKRSERFYECCKEPYPDVTFTVTMRRRTLYY

In some cases, a modified LGIC subunit described herein can include aIPD from a 5HT3 receptor. Examples of 5HT3 IPDs include, withoutlimitation, a murine 5HT3 IPD having the amino acid sequence set forthin SEQ ID NO:3, and a human 5HT3 IPD having the amino acid sequence setforth in SEQ ID NO:4. In some cases, a 5HT3 IPD can be a homolog,orthologue, or paralog of a 5HT3 IPD set forth in SEQ ID NO:3 or SEQ IDNO:4. In some cases, a 5HT3 IPD can be have at least 75 percent sequenceidentity (e.g., at least 80%, at least 82%, at least 85%, at least 88%,at least 90%, at least 93%, at least 95%, at least 97% or at least 99%sequence identity) to SEQ ID NO:3 of SEQ ID NO:4.

SEQ ID NO: 3 IIRRRPLFYAVSLLLPSIFLMVVDIVGFCLPPDSGERVSFKITLLLGYSVFLIIVSDTLPATIGTPLIGVYFVVCMALLVISLAETIFIVRLVHKQDLQRPVPDWLRHLVLDRIAWILCLGEQPMAHRPPATFQANKTDDCSGSDLLPAMGNHCSHVGGPQDLEKTPRGRGSPLPPPREASLAVRGLLQELSSIRHFLEKRDEMREVARDWLRVGYVLDRLLFRIYLLAVLAYSITLVTLWSIWHYS SEQ ID NO: 4IIRRRPLFYVVSLLLPSIFLMVMDIVGFYLPPNSGERVSFKITLLLGYSVFLIIVSDTLPATAIGTPLIGVYFVVCMALLVISLAETIFIVRLVHKQDLQQPVPAWLRHLVLERIAWLLCLREQSTSQRPPATSQATKTDDCSAMGNHCSHMGGPQDFEKSPRDRCSPPPPPREASLAVCGLLQELSSIRQFLEKRDEIREVARDWLRVGSVLDKLLFHIYLLAVLAYSITLVMLWSIWQYA

In some cases, a modified LGIC subunit described herein can include anIPD from a GlyR. Examples of GlyR IPDs include, without limitation, amurine GlyR IPD having the amino acid sequence set forth in SEQ ID NO:5.In some cases, a GlyR IPD can be a homolog, orthologue, or paralog ofthe human GlyR IPD set forth in SEQ ID NO:5. In some cases, a GlyR IPDcan be have at least 75 percent sequence identity (e.g., at least 80%,at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, atleast 95%, at least 97% or at least 99% sequence identity) to SEQ IDNO:5.

SEQ ID NO: 5 MGYYLIQMYIPSLLIVILSWISFWINMDAAPARVGLGITTVLTMTTQSSGSRASLPKVSYVKAIDIWMAVCLLFVFSALLEYAAVNFVSRQHKELLRFRRKRRHHKEDEAGEGRFNFSAYGMGPACLQAKDGISVKGANNSNTTNPPPAPSKSPEEMRKLFIQRAKKIDKISRIGFPMAFLIFNMFYWIIYKIVRREDVHNQ

In some cases, a modified LGIC subunit described herein can include anIPD from a GABA receptor (e.g., GABA_(A)-ρ, also referred to asGABA_(C)). Examples of GABA_(A)-p IPDs include, without limitation, ahuman GABA_(A)-ρ IPD having the amino acid sequence set forth in SEQ IDNO:9. In some cases, a GABA_(A)-ρ IPD can be a homolog, orthologue, orparalog of the human GABA_(A)-ρ IPD set forth in SEQ ID NO:9. In somecases, a GABA_(A)-ρ IPD can be have at least 75 percent sequenceidentity (e.g., at least 80%, at least 82%, at least 85%, at least 88%,at least 90%, at least 93%, at least 95%, at least 97% or at least 99%sequence identity) to SEQ ID NO:9.

SEQ ID NO: 9 LLQTYFPATLMVMLSWVSFWIDRRAVPARVPLGITTVLTMSTIITGVNASMPRVSYIKAVDIYLWVSFVFVFLSVLEYAAVNYLTTVQERKEQKLREKLPCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPENGEKPDRMMVQLTLASERSSPQRKSQRSSYVSMRIDTHAIDKYSRIIFPAAYILFNLIYWSIFS

In calculating percent sequence identity, two sequences are aligned andthe number of identical matches of amino acid residues between the twosequences is determined. The number of identical matches is divided bythe length of the aligned region (i.e., the number of aligned amino acidresidues) and multiplied by 100 to arrive at a percent sequence identityvalue. It will be appreciated that the length of the aligned region canbe a portion of one or both sequences up to the full-length size of theshortest sequence. It also will be appreciated that a single sequencecan align with more than one other sequence and hence, can havedifferent percent sequence identity values over each aligned region. Thealignment of two or more sequences to determine percent sequenceidentity can be performed using the computer program ClustalW anddefault parameters, which calculates the best match between a query andone or more subject sequences, and aligns them so that identities,similarities and differences can be determined. See, e.g., Chenna etal., 2003, Nucleic Acids Res., 31(13):3497-500.

In cases where a modified LGIC subunit described herein is a chimericLGIC subunit, the chimeric LGIC subunit can include a LBD and IPD fromthe same species or a LBD and IPD from different species. In some cases,a chimeric LGIC subunit can include a LBD from a human LGIC protein andan IPD from a human LGIC protein. For example, a chimeric LGIC subunitcan include a human α7 LBD and a human GlyR IPD. In some cases, achimeric LGIC subunit can include a LBD from a human LGIC protein and anIPD from a murine LGIC protein. For example, a chimeric LGIC subunit caninclude a human 07 LBD and a murine 5HT3 IPD.

In cases where a modified LGIC subunit described herein is a chimericLGIC subunit, the chimeric LGIC subunit can include varied fusion pointsconnecting the LBD and the IPD such that the number of amino acids in aLBD may vary when the LBD is fused with different IPDs to form achimeric channel subunit. For example, the length of an α7 nAChR LBDused to form a chimeric LGIC subunit with a 5HTS IPD is different fromthe length of an α7 nAChR LBD used to form a chimeric LGIC subunit witha GlyR IPD (compare, for example, FIGS. 1A and 1C to FIG. 1B).

A modified LGIC subunit described herein can include a LBD having atleast one modified amino acid and/or an IPD having at least one modifiedamino acid. For example, a modified LGIC subunit described herein caninclude a α7 LBD having at least 75 percent sequence identity to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and an amino acidsubstitution at amino acid residue 27, 41, 77, 79, 131, 139, 141, 175,210, 216, 217, and/or 219. For example, a modified LGIC subunitdescribed herein can include a GlyR IPD having at least 75 percentsequence identity to a sequence set forth in SEQ ID NO:5, and an aminoacid substitution at amino acid residue 298 of an α7-GlyR chimericreceptor (e.g., SEQ ID NO:7). For example, a modified LGIC subunitdescribed herein can include a GABA_(C) IPD having at least 75 percentsequence identity to SEQ ID NO:9, and an amino acid substitution atamino acid residue 298 of an α7-GABA_(C) chimeric receptor (e.g., SEQ IDNO:10). In some cases, a modified LGIC subunit described herein caninclude more than one (e.g., two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, or more) amino acid modifications. Themodification can be an amino acid substitution. In some cases, themodified amino acid can confer pharmacological selectivity to themodified LGIC. For example, the modified amino acid can confer themodified LGIC with selective binding of an exogenous LGIC ligand. Forexample, the modified amino acid can confer the modified LGIC withreduced (minimized or eliminated) binding of an unmodified LGIC subunit(an LGIC subunit lacking the modification and/or an endogenous LGICsubunit). For example, the modified amino acid can confer the modifiedLGIC with reduced (minimized or eliminated) binding of an endogenousLGIC ligand.

In some aspects, a modified LGIC subunit described herein can include atleast one modified amino acid that confers the modified LGIC withselective binding (e.g., enhanced binding or increased potency) with anexogenous LGIC ligand. The binding with an exogenous LGIC ligand can beselective over the binding with an endogenous LGIC ligand. A modifiedLGIC subunit with selective binding with an exogenous LGIC ligand caninclude any appropriate LDB (e.g., a α7 LBD). In some aspects, themodified LGIC subunit can include a α7 LBD set forth in SEQ ID NO:1, SEQID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modificationcan be a substitution at amino acid residue 77, 79, 131 139, 141, 175,and/or 216. In some cases, the tryptophan at amino acid residue 77 ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can besubstituted with a hydrophobic amino acid residue such as phenylalanine(e.g., W77F), tyrosine (e.g., W77Y), or methionine (e.g., W77M). Forexample, a modified LGIC subunit described herein can include a α7 LBDset forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 andhaving a W77F substitution. In some cases, the glutamine at amino acidresidue 79 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12can be substituted with an amino acid residue such as alanine (e.g.,Q79A), glycine (e.g., Q79G), or serine (e.g., Q79S). For example, amodified LGIC subunit described herein can include a α7 LBD having aQ79G substitution. In some cases, the leucine at amino acid residue 131of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can besubstituted with an amino acid residue such as alanine (e.g., L131A),glycine (e.g., L131G), methionine (e.g., L131M), asparagine (e.g.,L131N), glutamine (e.g., L131Q), valine (e.g., L131V), or phenylalanine(e.g., L131F). In some cases, the glycine at amino acid residue 175 ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can besubstituted with an amino acid residue such as lysine (e.g., G175K),alanine (e.g., G175A), phenyalanine (e.g., G175F), histidine (e.g.,G175H), methionine (e.g., G175m), arginine (e.g., G175R), serine (e.g.,G175S), valine (e.g., G175V). In some cases, the proline at amino acidresidue 216 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12can be substituted with an amino acid residue such as isoleucine (e.g.,P216I). A modified LGIC subunit with selective binding with an exogenousLGIC ligand can include any appropriate IPD (e.g., a GlyR IPD or aGABA_(A)-ρ IPD). In some aspects, the modified LGIC subunit can includea GlyR IPD set forth in SEQ ID NO:5, and the amino acid modification canbe a substitution at amino acid residue 298 of an α7-GlyR chimericreceptor (e.g., SEQ ID NO:7). In some cases, the alanine at amino acidresidue 298 of SEQ ID NO:7 can be substituted with an amino acid residuesuch as glycine (e.g., A298G). In some aspects, the modified LGICsubunit can include the a GABA_(A)-ρ IPD set forth in SEQ ID NO:9, andthe amino acid modification can be a substitution at amino acid residue298 of an α7-GABA_(A)-ρ chimeric receptor (e.g., SEQ ID NO:10). In somecases, the tryptophan at amino acid residue 298 of SEQ ID NO:10 can besubstituted with an amino acid residue such as alanine (e.g., W298A).

In some cases, a modified LGIC subunit described herein can include morethan one (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, or more) amino acid modifications. For example, amodified LGIC subunit described herein can have at least 75 percentsequence identity to SEQ ID NO:7 and can include a Q79G substitution anda A298G substitution. Additional examples of modifications that canconfer the modified LGIC with selective binding of an exogenous LGICligand include modifications described elsewhere (see, e.g., U.S. Pat.No. 8,435,762).

A modified LGIC subunit that selectively binds (e.g., enhanced bindingor increased potency) an exogenous LGIC ligand over an endogenous (e.g.,a canonical) LGIC ligand can also be described as having enhancedpotency for an exogenous ligand. In some cases, a modified LGIC subunitdescribed herein that selectively binds an exogenous LGIC ligand canhave at least 4 fold (e.g., at least 5 fold, at least 6 fold, at least 7fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19fold, or at least 20 fold) enhanced potency for an exogenous ligand. Insome cases, a modified LGIC subunit described herein that selectivelybinds an exogenous LGIC ligand can have about 4 fold to about 200 fold(e.g., about 4 fold to about 200 fold, about 5 fold to about 180 fold,about 6 fold to about 175 fold, about 7 fold to about 150 fold, about 8fold to about 125 fold, about 9 fold to about 100 fold, about 10 fold toabout 90 fold, about 11 fold to about 75 fold, about 12 fold to about 65fold, about 13 fold to about 50 fold, about 14 fold to about 40 fold, orabout 15 fold to about 30 fold) enhanced potency for an exogenousligand. For example, a modified LGIC subunit described herein thatselectively binds an exogenous LGIC ligand can have about 10 fold toabout 100 fold enhanced potency for an exogenous ligand. For example, amodified LGIC subunit described herein that selectively binds anexogenous LGIC ligand can have about 10 fold to about 20 fold enhancedpotency for an exogenous ligand.

In some aspects, a modified LGIC subunit described herein can include atleast one modified amino acid that confers the modified LGIC withreduced (e.g., minimized or eliminated) binding with an unmodified LGICsubunit. The binding with a modified LGIC subunit having the samemodification can be selective over the binding with an unmodified LGICsubunit. An unmodified LGIC subunit can be a LGIC subunit lacking themodification that confers the modified LGIC with reduced binding with anunmodified LGIC subunit or an unmodified LGIC can be an endogenous LGICsubunit. The modification that confers the modified LGIC with reducedbinding with an unmodified LGIC subunit can be a charge reversalmodification. A modified LGIC subunit with reduced binding with anunmodified LGIC subunit can include any appropriate LBD (e.g., a α7LBD). In some aspects, the modified LGIC subunit can include a α7 LBDset forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12,and the amino acid modification can be a substitution at amino acidresidue 27 and/or 41. For example, the arginine at amino acid residue 27of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can besubstituted with an aspartic acid (e.g., R27D). For example, theglutamic acid at amino acid residue 41 of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:11, or SEQ ID NO:12 can be substituted with an arginine (e.g.,E41R). In some cases, a modified LGIC subunit described herein caninclude a α7 LBD having a R27D substitution and a E41R.

In some aspects, a modified LGIC subunit described herein can include atleast one modified amino acid that confers the modified LGIC withreduced (e.g., minimized or eliminated) binding of an endogenous LGICligand. The endogenous LGIC ligand can be ACh. A modified LGIC subunitwith reduced binding of an endogenous LGIC ligand can include anyappropriate IPD (e.g., a GlyR LBD). For example, the modified LGICsubunit can include a α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQID NO:11, or SEQ ID NO:12, and the amino acid modification can be asubstitution at amino acid residue 115, 131, 139, 210, 217 and/or 219.In some cases, the tyrosine at amino acid residue 115 of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with aphenylalanine (e.g., Y115F). In some cases, the leucine at amino acidresidue 131 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12can be substituted with an amino acid residue such as alanine (e.g.,L131A), glycine (e.g., L131G), methionine (e.g., L131M), asparagine(e.g., L131N), glutamine (e.g., L131Q), valine (e.g., L131V), orphenylalanine (e.g., L131F). In some cases, the glutamine at amino acidresidue 139 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12can be substituted with a glycine (e.g., Q139G) or a leucine (e.g.,Q139L). In some cases, the tyrosine at amino acid residue 210 of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted witha phenylalanine (e.g., Y210F). In some cases, the tyrosine at amino acidresidue 217 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12can be substituted with a phenylalanine (e.g., Y217F). In some cases,the aspartate at amino acid residue 219 of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:11, or SEQ ID NO:12 can be substituted with an alanine (e.g.,D219A).

In some aspects, a modified LGIC subunit described herein can include atleast one modified amino acid that confers the modified LGIC withincreased ion conductance. In some cases, the modified LGIC subunit caninclude a 5HT3 IPD set forth in SEQ ID NO:3, and the amino acidmodification can be a substitution at amino acid residue 425, 429,and/or 433. For example, a modified LGIC subunit described herein caninclude a 5HT3 IPD having a R425Q substitution, a R429D substitution,and a R433A substitution. In some cases, the modified LGIC subunit caninclude a 5HT3 IPD set forth in SEQ ID NO:4, and the amino acidmodification can be a substitution at amino acid residue 420, 424,and/or 428. For example, a modified LGIC subunit described herein caninclude a 5HT3 IPD having a R420Q substitution, a R424D substitution,and a R428A substitution.

In some cases, a modified LGIC described herein can include at least onechimeric α7-5HT3 LGIC subunit (SEQ ID NO:6) having a human α7 nAChR LBD(SEQ ID NO:1) with a Q79G amino acid substitution and a Y115F amino acidsubstitution, and a murine 5HT3 IPD (SEQ ID NO:3).

In some cases, a modified LGIC described herein can include at least onechimeric α7-5HT3 LGIC subunit (SEQ ID NO:6) having a human α7 nAChR LBD(SEQ ID NO:1) with a Q79G amino acid substitution and a Q139G amino acidsubstitution, and a murine 5HT3 IPD (SEQ ID NO:3).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a Q79G amino acid substitution and a Y115F amino acidsubstitution, and a human GlyR IPD (SEQ ID NO:5) with a A298G amino acidsubstitution.

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a Q79G amino acid substitution and a Q139G amino acidsubstitution, and a human GlyR IPD (SEQ ID NO:5) with a A298G amino acidsubstitution.

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a R27D amino acid substitution, a E41R amino acidsubstitution, a Q79G amino acid substitution, and a Y115F amino acidsubstitution, and a human GlyR IPD (SEQ ID NO:5) with a A298G amino acidsubstitution.

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residue 131 (e.g.,L131G, L131A, L131M, or L131N), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residues 131 (e.g.,L131G, L131A, L131M, or L131N) and Y115 (e.g., Y115F), and a human GlyRIPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residues 131 (e.g.,L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L), and a human GlyRIPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residues 131 (e.g.,L131G, L131A, L131M, or L131N) and 217 (e.g., Y217F), and a human GlyRIPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residues 131 (e.g.,L131G, L131A, L131M, or L131N), 139 (e.g., Q139L), and 217 (e.g.,Y217F), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO:2)with a substitution at amino acid residue 131 (e.g., L131G, L131A,L131M, or L131N), and a human 5HT3 IPD (SEQ ID NO:4).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residue 175 (e.g.,G175K), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO:2)with a substitution at amino acid residue 131 (e.g., L131G, L131A,L131M, or L131N) and 139 (e.g., Q139L), and a human 5HT3 IPD (SEQ IDNO:4) with a R420Q substitution, a R424D substitution, and a R428Asubstitution.

In some cases, a modified LGIC described herein can include at least onechimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO:2)with a substitution at amino acid residue 131 (e.g., L131G, L131A,L131M, or L131N) and 139 (e.g., Q139L) and 217 (e.g., Y217F), and ahuman 5HT3 IPD (SEQ ID NO:4) with a R420Q substitution, a R424Dsubstitution, and a R428A substitution.

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residues 175 (e.g.,G175K) and 115 (e.g., Y115F), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residues 175 (e.g.,G175K) and 115 (e.g., Y115F) and 79 (e.g., Q79G), and a human GlyR IPD(SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residues 175 (e.g.,G175K) and 77 (e.g., W77F) and 79 (e.g., Q79G), and a human GlyR IPD(SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residue 216 (e.g.,P216I), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:7) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residues 216 (e.g.,P216I) and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least onechimeric α7-GlyR LGIC subunit (SEQ ID NO:10) having a human α7 nAChR LBD(SEQ ID NO:2) with a substitution at amino acid residue 131 (e.g.,L131A, L131G, L131M, L131N, L131Q, L131V, or L131F), and a humanGABA_(C) IPD (SEQ ID NO:9).

In cases where a LBD and/or a IPD is a homolog, orthologue, or paralogof a sequence set forth herein (e.g., SEQ ID NOs:1-5 and/or 9), it isunderstood that reference to a particular modified amino acid residuecan shift to the corresponding amino acid in the homolog, orthologue, orparalog. For example, residues 425, 429, and 433 in a murine 5HT3 IPDset forth in SEQ ID NO:3 correspond to residues 420, 424, and 428 in ahuman 5HT3 IPD set forth in SEQ ID NO:4, and the R425Q, R429D, and R433Asubstitutions in a murine 5HT3 IPD correspond to R420Q, R424D, and R428Asubstitutions in a human 5HT3 IPD.

Any method can be used to obtain a modified LGIC subunit describedherein. In some cases, peptide synthesis methods can be used to make amodified LGIC subunit described herein. Examples of methods of peptidesynthesis include, without limitation, liquid-phase peptide synthesis,and solid-phase peptide synthesis. In some cases, protein biosynthesismethods can be used to make a modified LGIC subunit described herein.Examples of methods of protein biosynthesis include, without limitation,transcription and/or translation of nucleic acids encoding aphosphorylation-mimicking peptide provided herein. Similar modified LGICsubunits (e.g., modified subunits having essentially the samemodifications and/or having essentially the same amino acid sequence)will self-assemble through interactions between the LBDs to form amodified LGIC.

This document also provides nucleic acids encoding modified LGICsubunits described herein as well as constructs (e.g., plasmids,non-viral vectors, viral vectors (such as adeno-associated virus, aherpes simplex virus, or lentivirus vectors)) for expressing nucleicacids encoding modified LGIC subunits described herein. Nucleic acidsencoding modified LGIC subunits described herein can be operably linkedto any appropriate promoter. A promoter can be a native (i.e., minimal)promoter or a composite promoter. A promoter can be a ubiquitous (i.e.,constitutive) promoter or a regulated promoter (e.g., inducible, tissuespecific, cell-type specific (e.g., neuron specific, muscle specific,glial specific), and neural subtype-specific). Examples of promotersthat can be used to drive expression of nucleic acids encoding modifiedLGIC subunits described herein include, without limitation, synapsin,CAMKII, CMV, CAG enolase, TRPV1, POMC, NPY, AGRP, MCH, and Orexinpromoters. In some cases, a nucleic acid encoding a modified LGICsubunit described herein can be operably linked to a neuron specificpromoter.

This document also provides cells (e.g., mammalian cells) having amodified LGIC described herein. Mammalian cells having a modified LGICdescribed herein can be obtained by any appropriate method. In somecases, a pre-assembled modified LGIC can be provided to the cell. Insome cases, a nucleic acid encoding a modified LGIC subunit describedherein can be provided to the cell under conditions in which a modifiedLGIC subunit is translated and under conditions in which multiple (e.g.,three, four, five, six, or more) modified LGIC subunits can assembleinto a modified LGIC described herein.

LGIC Ligands

This document also provides LGIC ligands that can bind to and activatemodified LGICs described herein. A LGIC ligand that can bind to andactivate modified LGICs described herein can be exogenous or endogenous.A LGIC ligand that can bind to and activate modified LGICs describedherein can be naturally occurring or synthetic. A LGIC ligand that canbind to and activate modified LGICs described herein can be canonical ornon-canonical. A LGIC ligand that can bind to and activate modifiedLGICs described herein can be an agonist or an antagonist. In somecases, an LGIC ligand is an exogenous LGIC agonist. Examples of LGICligands include, without limitation, ACh, nicotine, epibatatine,cytisine, RS56812, tropisetron, nortropisetron, PNU-282987, PHA-543613,compound 0353, compound 0354, compound 0436, compound 0676, compound702, compound 723, compound 725, granisetron, ivermectin, mequitazine,promazine, varenicline, compound 765, compound 770,3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide,compound 773, and compound 774 (see, e.g., FIG. 3B, FIG. 5C, FIG. 10A,and FIG. 10B).

A LGIC ligand that can bind to and activate modified LGICs describedherein can have selective binding (e.g., enhanced binding or increasedpotency) for a modified LGIC described herein. In some cases, a LGICligand that can bind to and activate modified LGICs described hereindoes not bind to and activate endogenous receptors. A LGIC ligand thatselectively binds to and activates a modified LGIC (e.g., a modifiedLGIC having at least one amino acid modification that conferspharmacological selectivity to the modified LGIC) described herein overan unmodified LGIC ligand can also be described as having enhancedpotency for a modified LGIC. In some cases, a modified LGIC subunitdescribed herein that selectively binds an exogenous LGIC ligand canhave at least 5 fold (e.g., at least 10 fold, at least 15 fold, at least20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least80 fold, at least 85 fold, at least 95 fold, at least 100 fold, at least125 fold, at least 150 fold, at least 200 fold, at least 250 fold, or atleast 300 fold) enhanced potency for a modified LGIC. For example, aLGIC ligand that selectively binds to and activates a modified LGIC canhave about 10 fold to about 300 fold (e.g., about 10 fold to about 250fold, about 10 fold to about 200 fold, about 10 fold to about 150 fold,about 10 fold to about 100 fold, about 25 fold to about 300 fold, about50 fold to about 300 fold, about 100 fold to about 300 fold, about 200fold to about 300 fold, about 25 fold to about 250 fold, about 50 foldto about 200 fold, or about 100 fold to about 150 fold) enhanced potencyfor a modified LGIC. In some cases, a LGIC ligand that binds to andactivates a modified LGIC described herein can have a ligand potency ofless than 25 nM (e.g., less than 22 nM, less than 20 nM, less than 17nM, less than 15 nM, less than 13 nM, less than 12 nM, less than 11 nM,less than 10 nM, less than 5 nM, less, than 2 nM, or less than 1 nM).For example, a LGIC ligand that binds to and activates a modified LGICdescribed herein can have a ligand potency of less than 15 nM. In somecases, a LGIC ligand can have an EC50 of less than 25 nM (e.g., lessthan 22 nM, less than 20 nM, less than 17 nM, less than 15 nM, less than13 nM, less than 12 nM, less than 11 nM, or less than 10 nM) for amodified LGIC subunit described herein. For example, a LGIC ligand(e.g., tropisetron) can have an EC50 of about 11 nM for a modified LGICsubunit described herein (e.g., α7^(Q79G)-GlyR^(A298G)). For example, aLGIC ligand (e.g., nortropisetron) can have an EC50 of about 13 nM for amodified LGIC subunit described herein (e.g.,α7^(Q79G,Y115F)-GlyR^(A298G)). In some cases, a LGIC ligand can have anEC50 of greater than 20 μM (e.g., greater than 22 μM, greater than 25μM, greater than 35 μM, greater than 50, greater than 65 μM, greaterthan 80 μM, or greater than 100 μM) for a modified LGIC subunitdescribed herein. For example, a LGIC ligand (e.g., ACh) can have anEC50 of greater than 100 μM for a modified LGIC subunit described herein(e.g., α7^(Q79G,Y115F)-GlyR^(A298G)).

In some aspects, a LGIC ligand can be a synthetic ligand that can bindto and activate modified LGICs described herein can be a quinuclidine, atropane, a 9-azabicyclo[3.3.1]nonane, or a2-phenyl-7,8,9,10-tetrahydro-6H-6,10-methanoazepino[4,5-g]quinoxaline.

A LGIC ligand that can be to and activate a modified LGIC describedherein can have Formula I:

where X1 and X2 can independently be CH, CH2, O, NH, or NMe; each n canindependently be 0 or 1; Y can be O or S; A can be an aromaticsubstituent; and R can be H or pyridinymethylene. Examples of aromaticsubstituents include, without limitation, 4-chloro-benzene, 1H-indole,4-(trifluoromethyl) benzene, 4-chloro benzene, 2,5-dimethoxy benzene,4-chloroaniline, aniline, 5-(trifluoromethyl) pyridin-2-yl,6-(trifluoromethyl) nicotinic, and 4-chloro-benzene.

A LGIC ligand that can bind to and activate a modified LGIC describedherein can be a quinuclidine. A quinuclidine can have the structure ofFormula II:

where X3 can be O, NH, or CH2; Y can be O or S; A can be an aromaticsubstituent; and R can be H or pyridinylmethylene. Examples of aromaticsubstituents include without limitation, 1H-indole, 4-(trifluoromethyl)benzene, 4-chloro benzene, 2,5-dimethoxy benzene, 4-(trifluoromethyl)benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl) pyridin-2-yl,6-(trifluoromethyl) nicotinic, 3-chloro-4-fluoro benzene,4-chloro-benzene, and 1H-indole. Examples of quinuclidines include,without limitation, compounds PNU-282987, PHA-543613, 0456, 0434, 0436,0354, 0353, 0295, 0296, and 0676 (see, e.g., FIG. 5C, Table 3, and Table6).

A LGIC ligand that can bind to and activate a modified LGIC describedherein can be a tropane. A tropane can have the structure of FormulaIII:

where X2 can be NH or NMe; X3 can be O, NH, or CH2; Y can be O or S; andA can be an aromatic substituent. Example of aromatic substituentsinclude, without limitation, 1H-indole, 7-methoxy-1H-indole,7-methyl-1H-indole, 5-chloro-1H-indole, and 1H-indazole. Examples oftropanes include, without limitation, tropisetron, pseudo-tropisetron,nortropisetron, compound 737, and compound 745 (see, e.g., FIG. 5C,Table 3, and Table 6).

A LGIC ligand that can bind to and activate a modified LGIC describedherein can be a 9-azabicyclo[3.3.1]nonane. A 9-azabicyclo[3.3.1]nonanecan have the structure of Formula IV:

where X1 can be CH, X2 can be NH or NMe, X3 can be O, NH, or CH; Y canbe O or S; and A can be an aromatic substituent. An example of anaromatic substituent is, without limitation, 4-chloro-benzene. Examplesof 9-azabicyclo[3.3.1]nonanes include, without limitation, compound0536, compound 0749, compound 0751, compound 0760, and compound 0763(see, e.g., FIG. 5C, Table 3, and Table 6).

In some cases, a LGIC ligand can be an a6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine and canhave a structure shown in Formula V:

where R=H or CH3; and where A=H or an aromatic substituent. Examples of6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepines include,without limitation, varenicline, compound 0765, and compound 0770 (see,e.g., FIG. 10A, Table 3, and Table 9).

In some cases, a LGIC ligand can be a 1,4-diazabicyclo[3.2.2]nonane andcan have a structure shown in Formula VI:

where R=H, F, NO₂. Examples of 1,4-diazabicyclo[3.2.2]nonanes include,without limitation,3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide,compound 0773, and compound 0774 (see, e.g., FIG. 10B, Table 6, andTable 9).

Methods of Using

This document also provides methods of using a modified LGIC describedherein and a LGIC ligand that can bind to and activate the modified LGICas described herein. A LGIC ligand that can bind to and activate themodified LGIC can be used to activate a modified LGIC with temporaland/or spatial control based on delivery of the ligand.

In some aspects, a modified LGIC described herein and a LGIC ligand thatcan bind to and activate the modified LGIC as described herein can beused to identify a ligand that selectively binds to a modified LGICdescribed herein. For example, such screening methods can includeproviding one or more candidate ligands to a modified LGIC describedherein, and detecting binding between the candidate ligand and themodified LGIC.

Any appropriate method can be used to detect binding between a candidateligand and the modified LGIC and any appropriate method can be used todetect activity of a modified LGIC. For example, the ability of a ligandto bind to and activate a modified LGIC can be measured by assaysincluding, but not limited to, membrane potential (MP) assay (e.g., afluorescence MP assay), radioactive binding assays, and/or voltage clampmeasurement of peak currents and sustained currents.

In some aspects, a modified LGIC described herein and a LGIC ligand thatcan bind to and activate the modified LGIC as described herein can beused to treat a mammal having a channelopathy (e.g., a neuralchannelopathy or a muscle channelopathy). For example, a mammal having achannelopathy can be treated by administering a modified LGIC describedherein, and then administering a LGIC ligand that can bind to andactivate the modified LGIC. For example, a mammal having a channelopathycan be treated by administering a modified LGIC described herein (e.g.,including at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO:6)having a human α7 nAChR LBD (SEQ ID NO:2) with a R27D amino acidsubstitution, a E41R amino acid substitution, a Q79G amino acidsubstitution, and a Y115F amino acid substitution, and a human GlyR IPD(SEQ ID NO:5) with a A298G amino acid substitution), and thenadministering tropisetron. For example, a mammal having a channelopathycan be treated by administering a modified LGIC described hereinincluding a modified human α7 nAChR LBD (e.g., SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:11, or SEQ ID NO:12) with an L131 amino acid substitution(e.g., L131G, L131A, L131M, or L131N) and, optionally, a Q79S amino acidsubstitution, a Q139L amino acid substitution, and/or a Y217F amino acidsubstitution, and then administering varenicline, tropisetron, and/orcompound 765.

Any type of mammal can be treated using a modified LGIC described hereinand a LGIC ligand that can bind to and activate the modified LGIC asdescribed herein. For example, humans and other primates such as monkeyscan be treated using a modified LGIC described herein and a LGIC ligandthat can bind to and activate the modified LGIC as described herein. Insome cases, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, andrats can be treated using a modified LGIC described herein and a LGICligand that can bind to and activate the modified LGIC as describedherein.

Any appropriate method can be used to identify a mammal having achannelopathy and/or a mammal at risk of developing a channelopathy. Forexample, genetic testing can be used to identify a mammal having achannelopathy and/or a mammal at risk of developing a channelopathy.

Once identified as having a channelopathy and/or a mammal at risk ofdeveloping a channelopathy, the mammal can be administered or instructedto self-administer a modified LGIC described herein, and thenadministered or instructed to self-administer a LGIC ligand that canbind to and activate the modified LGIC as described herein. A modifiedLGIC described herein and a LGIC ligand that can bind to and activatethe modified LGIC as described herein can be administered together orcan be administered separately.

When treating a mammal having a channelopathy and/or a mammal at risk ofdeveloping a channelopathy using the materials and methods describedherein, the channelopathy can be any channelopathy. As used herein, achannelopathy can be any disease or disorder caused by aberrant ionchannel function and/or aberrant ligand function, or which could bealleviated by modulated ion channel function and/or altered cellular ionflux (e.g., calcium ion flux). A channelopathy can be congenital oracquired. Examples of channelopathies include, without limitation,Bartter syndrome, Brugada syndrome, catecholaminergic polymorphicventricular tachycardia (CPVT), congenital hyperinsulinism, cysticfibrosis, Dravet syndrome, episodic ataxia, erythromelalgia, generalizedepilepsy (e.g., with febrile seizures), familial hemiplegic migraine,fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodicparalysis, Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g.,Romano-Ward syndrome), short QT syndrome, malignant hyperthermia,mucolipidosis type IV, myasthenia gravis, myotonia congenital,neuromyelitis optica, neuromyotonia, nonsyndromic deafness, paramyotoniacongenital, retinitis pigmentosa, timothy syndrome, tinnitus, seizure,trigeminal neuralgia, and multiple sclerosis. Alternatively, or inaddition, the materials and methods described herein can be used inother applications including, without limitation, pain treatment, cancercell therapies, appetite control, spasticity treatment, muscle dystoniatreatment, tremor treatment, and movement disorder treatment.

In some cases, a modified LGIC described herein and a LGIC ligand thatcan bind to and activate the modified LGIC as described herein can beused to modulate the activity of a cell. The activity of the cell thatis modulated using a modified LGIC described herein and a LGIC ligandthat can bind to and activate the modified LGIC as described herein canbe any cellular activity. Examples of cellular activities include,without limitation, active transport (e.g., ion transport), passivetransport, excitation, inhibition, ion flux (e.g., calcium ion flux),and exocytosis. The cellular activity can be increased or decreased. Forexample, a modified LGIC described herein and a LGIC ligand that canbind to and activate the modified LGIC as described herein can be usedto modulate (e.g., increase) ion transport across the membrane of acell. For example, a modified LGIC described herein and a LGIC ligandthat can bind to and activate the modified LGIC as described herein canbe used to modulate (e.g., increase) the excitability of a cell.

A modified LGIC described herein and a LGIC ligand that can bind to andactivate the modified LGIC as described herein can be used to modulatethe activity of any type of cell in a mammal. The cell can be a neuron,a glial cell, a myocyte, an immune cell (e.g., neutrophils, eosinophils,basophils, lymphocytes, and monocytes), an endocrine cell, or a stemcell (e.g., an embryonic stem cell). In some cases, the cell can be anexcitable cell. The cell can be in vivo or ex vivo.

A modified LGIC described herein can be administered by any appropriatemethod. A modified LGIC can be administered as modified LGIC subunits oras pre-assembled modified LGICs. A modified LGIC can be administered asa nucleic acid encoding a modified LGIC. A modified LGIC can beadministered as a nucleic acid encoding a modified LGIC subunitdescribed herein. For example, a nucleic acid can be delivered as nakednucleic acid or using any appropriate vector (e.g., a recombinantvector). Vectors can be a DNA based vector, an RNA based, or combinationthereof. Vectors can express a nucleic acid in dividing cells ornon-dividing cells. Examples of recombinant vectors include, withoutlimitation, plasmids, viral vectors (e.g., retroviral vectors,adenoviral vectors, adeno-associated viral vectors, and herpes simplexvectors), cosmids, and artificial chromosomes (e.g., yeast artificialchromosomes or bacterial artificial chromosomes). In some cases, anucleic acid encoding a modified LGIC subunit described herein can beexpressed by an adeno-associated viral vector.

A modified LGIC described herein can be detected (e.g., to confirm itspresence in a cell) by any appropriate method. In some cases, an agentthat selectively binds a modified LGIC can be used to detect themodified LGIC. Examples of agents that can be used to bind to a modifiedLGIC described herein include, without limitation, antibodies, proteins(e.g., bungarotoxin), and small molecule ligands (e.g., PET ligands). Anagent that selectively binds a modified LGIC can include a detectablelabel (e.g., fluorescent labels, radioactive labels, positron emittinglabels, and enzymatic labels). Methods to detect LGIC expression in acell can include fluorescence imaging, autoradiography, functional MRI,PET, and SPECT.

A modified LGIC described herein and a LGIC ligand that can bind to andactivate the modified LGIC as described herein can be administered to amammal having a channelopathy and/or at risk of developing achannelopathy as a combination therapy with one or more additionalagents/therapies used to treat a channelopathy. For example, acombination therapy used to treat a mammal having a channelopathy asdescribed herein can include administering a modified LGIC describedherein and a LGIC ligand that can bind to and activate the modified LGICas described herein and treating with acetazolaminde, dichlorphenamide,mexilitine, glucose, calcium gluconate, L-DOPA, muscle stimulation,spinal stimulation, brain stimulation, and/or nerve stimulation.

In embodiments where a modified LGIC described herein and a LGIC ligandthat can bind to and activate the modified LGIC as described herein areused in combination with additional agents/therapies used to treat achannelopathy, the one or more additional agents can be administered atthe same time or independently. For example, a modified LGIC describedherein and a LGIC ligand that can bind to and activate the modified LGICas described herein first, and the one or more additional agentsadministered second, or vice versa. In embodiments where a modified LGICdescribed herein and a LGIC ligand that can bind to and activate themodified LGIC as described herein are used in combination with one ormore additional therapies used to treat a channelopathy, the one or moreadditional therapies can be performed at the same time or independentlyof the administration of a modified LGIC described herein and a LGICligand that can bind to and activate the modified LGIC as describedherein. For example, a modified LGIC described herein and a LGIC ligandthat can bind to and activate the modified LGIC as described herein canbe administered before, during, or after the one or more additionaltherapies are performed.

In some cases, a modified LGIC described herein and/or a LGIC ligandthat can bind to and activate the modified LGIC as described herein canbe formulated into a pharmaceutically acceptable composition foradministration to a mammal having a channelopathy or at risk ofdeveloping a channelopathy. For example, a therapeutically effectiveamount of a modified LGIC described herein (e.g., a nucleic acidencoding a modified LGIC described herein) and/or a LGIC ligand that canbind to and activate the modified LGIC as described herein can beformulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. A pharmaceutical composition canbe formulated for administration in solid or liquid form including,without limitation, sterile solutions, suspensions, sustained-releaseformulations, tablets, capsules, pills, powders, and granules.

Pharmaceutically acceptable carriers, fillers, and vehicles that may beused in a pharmaceutical composition described herein include, withoutlimitation, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A pharmaceutical composition containing a modified LGIC described hereinand/or a LGIC ligand that can bind to and activate the modified LGIC asdescribed herein can be designed for oral, parenteral (includingsubcutaneous, intracranial, intraarterial, intramuscular, intravenous,intracoronary, intradermal, or topical), or inhaled administration. Whenbeing administered orally, a pharmaceutical composition containing atherapeutically effective amount of a modified LGIC described herein(e.g., a nucleic acid encoding a modified LGIC described herein) and/ora LGIC ligand that can bind to and activate the modified LGIC asdescribed herein can be in the form of a pill, tablet, or capsule.Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions that can contain anti-oxidants,buffers, bacteriostats, and solutes which render the formulationisotonic with the blood of the intended recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents andthickening agents. Compositions for inhalation can be delivered using,for example, an inhaler, a nebulizer, and/or a dry powder inhaler. Theformulations can be presented in unit-dose or multi-dose containers, forexample, sealed ampules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules, and tablets.

A pharmaceutically acceptable composition including a therapeuticallyeffective amount of a modified LGIC described herein (e.g., a nucleicacid encoding a modified LGIC described herein) and/or a LGIC ligandthat can bind to and activate the modified LGIC as described herein canbe administered locally or systemically. In some cases, a compositioncontaining a therapeutically effective amount of a modified LGICdescribed herein (e.g., a nucleic acid encoding a modified LGICdescribed herein) and/or a LGIC ligand that can bind to and activate themodified LGIC as described herein can be administered systemically byvenous or oral administration to, or inhalation by a mammal (e.g., ahuman). In some cases, a composition containing a therapeuticallyeffective amount of a modified LGIC described herein (e.g., a nucleicacid encoding a modified LGIC described herein) and/or a LGIC ligandthat can bind to and activate the modified LGIC as described herein canbe administered locally by percutaneous, subcutaneous, intramuscular,intracranial, or open surgical administration (e.g., injection) to atarget tissue of a mammal (e.g., a human).

Effective doses can vary depending on the severity of the channelopathy,the route of administration, the age and general health condition of thesubject, excipient usage, the possibility of co-usage with othertherapeutic treatments such as use of other agents, and the judgment ofthe treating physician.

The frequency of administration can be any frequency that improvessymptoms of a channelopathy without producing significant toxicity tothe mammal. For example, the frequency of administration can be fromabout once a week to about three times a day, from about twice a monthto about six times a day, or from about twice a week to about once aday. The frequency of administration can remain constant or can bevariable during the duration of treatment. A course of treatment with acomposition containing a therapeutically effective amount of a modifiedLGIC described herein (e.g., a nucleic acid encoding a modified LGICdescribed herein) and/or a LGIC ligand that can bind to and activate themodified LGIC as described herein can include rest periods. For example,a composition containing a therapeutically effective amount of amodified LGIC described herein (e.g., a nucleic acid encoding a modifiedLGIC described herein) and/or a LGIC ligand that can bind to andactivate the modified LGIC as described herein can be administered dailyover a two week period followed by a two week rest period, and such aregimen can be repeated multiple times. As with the effective amount,various factors can influence the actual frequency of administrationused for a particular application. For example, the effective amount,duration of treatment, use of multiple treatment agents, route ofadministration, and severity of the channelopathy may require anincrease or decrease in administration frequency.

An effective duration for administering a composition containing atherapeutically effective amount of a modified LGIC described herein(e.g., a nucleic acid encoding a modified LGIC described herein) and/ora LGIC ligand that can bind to and activate the modified LGIC asdescribed herein can be any duration that improves symptoms of achannelopathy without producing significant toxicity to the mammal. Forexample, the effective duration can vary from several days to severalweeks, months, or years. In some cases, the effective duration for thetreatment of a channelopathy can range in duration from about one monthto about 10 years. Multiple factors can influence the actual effectiveduration used for a particular treatment. For example, an effectiveduration can vary with the frequency of administration, effectiveamount, use of multiple treatment agents, route of administration, andseverity of the channelopathy being treated.

In certain instances, a course of treatment and the symptoms of themammal being treated for a channelopathy can be monitored. Anyappropriate method can be used to monitor the symptoms of achannelopathy.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: Potency-Enhancing Ligand Binding Domain Mutations

A screen was performed with a panel of 41 α7-5HT3 chimeric channelshaving mutant LBDs against a panel of 51 clinically used drugs withchemical similarity to nicotinic receptor agonists. Mutations were atresidues highlighted in FIG. 1. The screen revealed mutations at Gln⁷⁹in the α7 nAChR LBD that enhanced potency for the known nAChR agonisttropisetron (FIG. 2). These mutations (Q79A, Q79G, Q79S) reduce the sizeof the amino acid side chain. Some mutant ion channel-ligandcombinations gave up to 12-fold improvement in potency (Table 1, FIG.3). Canonical α7 nAChR agonists, ACh, nicotine, epibatidine, and theanti-smoking drug varenicline were not significantly affected by Q79A,Q79G, or Q79S mutations. However, a subset of α7 nAChR agonists showedenhanced potency with some of the mutations. Cytisine, RS56812,tropisetron, nortropisetron, and PNU-282987 showed significantlyimproved potency for α7^(Q79G)-5HT3. Additionally, nortropisetron andPNU-282987 showed a significantly enhanced potency for α7^(Q79A)-5HT3and α7^(Q79S)-5HT3, respectively. In general, agonists based on aquinuclidine or tropane pharmacophore with a linked aromatic structurethat interacts with the complementary binding face of the ligand bindingdomain showed improved potency with Gln79 substitution with the smalleramino acid residues Ala, Gly, or Ser. For most agonists, α7^(Q79G)-5HT3was the most preferred mutant chimeric ion channel.

TABLE 1 Potency of nAChR agonists against chimeric cation channelsmutated at Gln79 in HEK cells. Mean EC50, SEM in parentheses (μM).Agonist α7-5HT3 α7^(Q79A)-5HT3 α7^(Q79G)-5HT3 α7^(Q79S)-5HT3Acetylcholine 7.0 9.2 6.7 6.2 (0.8) (1.8) (0.6) (1.4) Nicotine 3.9 4.13.1 2.1 (0.4) (1.3) (0.5) (0.4) Epibatidine 0.053 0.067 0.050 0.044(0.006) (0.022) (0.008) (0.006) Varenicline 0.92 0.76 0.91 0.47 (0.16)(0.21) (0.12) (0.07) Cytisine 8.2 4.0 1.7 4.4 (0.3) (0.9) (0.2) (1.0)RS56812 10 6.8 1.4 5.7 (1.8) (1.9) (0.2) (0.8) Tropisetron 0.24 0.080.035 0.11 (0.03) (0.02) (0.002) (0.02) Nortropisetron 0.061 0.010 0.0060.019 (0.021) (0.002) (0.001) (0.007) PNU-282987 0.22 0.037 0.018 0.023(0.03) (0.009) (0.003) (0.004)

These mutated LBDs were used to generate α7-GlyR chimeric channelshaving enhanced potency for most of these ligands up to 6-fold (FIG.4A). Like mutations of α7-5HT3, these mutations at Gln79 did notsignificantly affect potency of ACh, nicotine, epibatidine, varenicline,or cytisine. However, tropisetron, nortropisetron, and RS56812 showedsignificantly enhanced potency for α7^(Q79G)-GlyR. Similar to LBDmutations for α7-5HT3, nortropisetron had significantly enhanced potencyfor α7^(Q79A)-GlyR, and PNU-282987 showed significantly enhanced potencyfor α7^(Q79S)-GlyR. For most agonists, α7^(Q79G)-GlyR was the mostpreferred mutant chimeric ion channel.

Another relationship that was observed in the small molecule screen wasthat mutations at Trp77 conferred agonist activity for the druggranisetron at the α7^(W77F)-5HT3 (EC50: 1.2 μM), μ7^(W77Y)-5HT3 (EC50:1.1 μM), and α7^(W77F)-GlyR (EC50: 0.66 μM) receptors. Granisetron is a5HT3 receptor antagonist granisetron, which does not activate α7-5HT3 orα7-GlyR.

These results show that mutation of Q79 (to A, G, or S) in the α7 nAChRLBD enhanced binding of known LGIC ligands to modified LGICs.

Example 2: Potency Enhancing Ion Pore Domain Mutations

α7-GlyR channels having IPD mutations previously established in fulllength glycine receptor channels (T258S and A288G, GlyR numbering;equivalent to T268S and A298G for α7-GlyR numbering) were examined forenhanced potency for the allosteric agonist ivermectin. Channels havingα7-GlyR^(T268S) were found to have substantial ligand-free openprobability, which rendered them unsuitable for ligand-controlledmanipulations of cells. Mutations at α7-GlyR^(A298G), which wereeffective for enhancing ivermectin potency at the full length glycinereceptor, led to modest change in open probability in the absence of theligand; thus this channel was examined for activity against a panel ofknown agonists. For canonical agonists ACh, nicotine, and epibatidine,as well as for varenicline and tropisetron, the agonist potency was notsignificantly enhanced in α7-GlyR^(A298G). A subset of α7 nAChR agonistsdid show up to a modest 4-fold increase in potency: RS56812, cytisine,PNU-282987, and nortropisetron were significantly more potent.Therefore, the effect of the IPD A298G mutation improved ligand potency,but depended on ligand structure and was not as effective as mutationsin the LBD.

The Q79G mutation in the LBD and the A298G IPD mutation for α7-GlyR wasexamined (Table 2). The double mutant chimeric channel,α7^(Q79G)-GlyR^(A298G), led to synergistic enhancement of potencyshowing up to 18-fold enhancement of potency relative to α7-GlyR to α7nAChR agonists. The enhancement from this double mutant channel wasgreater than that from the individual mutations for agonists RS56812,tropisetron, nortropisetron, and PNU-282987. Further underscoring theunexpected structural sensitivity of this combination of mutations,multiple agonists, such as ACh, nicotine, epibatidine, varenicline, andcytisine were not significantly changed between α7-GlyR andα7^(Q79G)-GlyR^(A298G). Therefore, combination of the LBD mutation Q79Gwith the IPD mutation A298G led to a synergistic effect where potencyfor some but not all nicotinic agonists was greatly increased by˜10-20-fold.

TABLE 2 Potency of nAChR agonists against mutated chimeric chloridechannels. Mean EC50 and SEM in parentheses (μM) for agonist activity inHEK cells expressing chimeric channels. α7^(Q79G)- Agonist α7 GlyRα7^(Q79A)-GlyR α7^(Q79G)-GlyR α7^(Q79S)-GlyR α7-GlyR^(A298G)GlyR^(A298G) Acetylcholine 6.4 (1.2) 7.6 (1.7) 7.1 (1.2) 4.5 (1.2) 6.4(1.8) 4.8 (0.5) Nicotine 5.0 (1.8) 2.6 (0.7) 4.1 (0.3) 1.4 (0.4) 3.1(1.8) 2.2 (0.6) Epibatidine 0.062 (0.021) 0.038 (0.005) 0.069 (0.011)0.024 (0.003) 0.018 (0.001) 0.032 (0.007) Varenicline 0.62 (0.2)  0.48(0.08)  1.1 (0.25) 0.28 (0.06) 0.25 (0.04) 0.33 (0.08) Cytisine 6.4(2.0) 4.5 (0.6) 5.6 (2.1) 2.5 (0.7)  2.1 (0.28) 2.8 (1.0) RS56812 6.5(1.8) 3.5 (0.5)  2.0 (0.15) 2.8 (0.5) 2.3 (0.1) 0.61 (0.14) Tropisetron 0.15 (0.045) 0.044 (0.008) 0.038 (0.003) 0.040 (0.009) 0.065 (0.026)0.011 (0.002) Nortropisetron 0.022 (0.007) 0.004 (0.001) 0.008 (0.003)0.005 (0.001) 0.005 (0.001) 0.002 (0.001) PNU-282987  0.13 (0.038) 0.022(0.004) 0.026 (0.005) 0.014 (0.002) 0.035 (0.005) 0.007 (0.001)

These results show that mutation of Q79 (to A, G, or S) in the α7 nAChRLBD and/or mutation of A298 (to G) in the GlyR IPD further enhancedselective binding of known LGIC ligands to modified LGICs.

Example 3: Molecules Exhibiting Enhanced Potency

Based on the structure activity relationship of known agonists thatshowed enhanced potency with α7^(Q79G)-GlyR^(A298G), a variety ofsynthetic molecules comprised of either quinuclidine, tropane, or9-azabicyclo[3.3.1]nonane pharmacophores with one or more aromatic sidechain substituents were tested. In addition, the known α7 nAChR agonistPHA-543613 (Walker et al 2006, Wishka et al 2006) was also tested andshowed exceptional potency for α7^(Q79G)-GlyR^(A298G). These moleculesgenerally showed enhanced potency 10-fold to 100-fold (Table 3),indicating that, for these pharmacophores, a range of structuralfeatures were compatible with improved potency forα7^(Q79G)-GlyR^(A298G).

These results show that modified LGICs can be activated by syntheticquinuclidine-containing and tropane-containing LGIC ligands.

TABLE 3 Potency of compounds against chimeric channels. Mean EC50 andSEM in parentheses (μM) for agonist activity in HEK cells expressingchimeric channels. Partial refers to partial agonist activity.α7^(Q79G)- C—X α7-5HT3 α7-GlyR GlyR^(A298G) Compound X₁ X₂ X₃ Y C₁ n C₂n C₃ n config R A EC₅₀ (μM) EC₅₀ (μM) EC₅₀ (μM) PNU-282987 N CH₂ NH O 01 0 R H 4-chloro-benzene 0.22 0.13 0.007 Tropisetron C NMe O O 1 0 0Endo H 1H-indole 0.24 0.15 0.011 Pseudo- C NMe O O 1 0 0 Exo H 1H-indole2 0.7 <0.2 tropisetron Nortropisetron C NH O O 1 0 0 Endo H 1H-indole0.061 0.022 0.002 PHA-543613 N CH₂ NH O 0 1 0 R H furo[2,3]pyridine0.046 0.039 0.004 0542 C NMe NH S 1 0 0 Endo H 1H-indole 3.8 0.58 0.0720026 N CH₂ O O 0 1 0 S H 4-(trifluoromethyl) — 13.7 1.43 benzene 0456 NCH₂ CH₂—NH S 0 1 0 mix H 4-chloro benzene — 2.8 0.47 0434 N CH₂ NH O 0 10 mix pyridin-3- 2,5-dimethoxy >10 >10 0.19 ylmethyl benzene 0436 N CH₂NH O 0 1 0 mix pyridin-3- 4-(trifluoromethyl) 0.84 0.31 0.006 ylmethylbenzene 0354 N CH₂ NH S 0 1 0 R H 4-chloroaniline 1.4 partial 1.0 0.030353 N CH₂ NH O 0 1 0 S H aniline 0.65 0.27 0.01 0295 N CH₂ NH O 0 1 0 SH 5-(trifluoromethyl) >100 >100 4.6 pyridin-2-yl) 0296 N CH₂ NH O 0 1 0S H 6-(trifluoromethyl) >100 — 0.45 nicotinic 0536 C NMe NH S 1 0 1 EndoH 4-chloro-benzene >33 >100 9.1 0676 N CH₂ NH O 0 1 0 S H 1H-indole 0.030.018 0.002

Example 4: Mutations that Reduce Acetylcholine Responsiveness

The α7 nAChR has relatively low sensitivity to ACh compared to othernAChR isoforms, and potency enhancing mutations for tropane andquinuclidine ligands did not substantially alter the potency ofacetylcholine at these channels. Thus, the chimeric channels werefurther modified to reduce acetylcholine responsiveness of thesechannels. Acetylcholine responsiveness was considerably reduced to morethan 100 μM in some cases with additional LBD mutations Y115F and Q139Gthat that only modestly reduced the potency of some agonists forα7^(Q79G,Y115F)-5HT3, α7^(Q79G,Q139G)-5HT3, α7^(Q79G,Q139G)-GlyR^(A298),α7^(Q79G,Y115F)-GlyR^(A298G). For example, α7^(Q79G,Y115F)-GlyR^(A298G)has an EC50 of 13 nM for nortropisetron and >100 μM for ACh (Table 4).

TABLE 4 Potency of nAChR agonists against mutated chimeric chloridechannels with low acetylcholine responsiveness. Mean EC50 and SEM inparentheses (μM) for activity in HEK cells expressing chimeric channels.α7^(Q79G,Y115F)- α7^(Q79G,Q139G)- α7^(Q79G,Y115F)_ α7^(Q79G,Q139G)_α7^(R27D,E41R,Q79G,Y115F)_ 5HT3 5HT3 GlyR^(A298G) GlyR^(A298G)GlyR^(A298G) Acetylcholine >100 36 (2) >100 73 (27) >100 Nicotine 34 (4)24 (4) 22 (3) 30 (8) 7.5 (1.3) Tropisetron 0.10 (0.12) 0.31 (0.06) 0.086(0.043) 0.26 (0.04) 0.035 (0.021) Nortropisetron 0.028 (0.005) 0.047(0.013) 0.013 (0.001) 0.031 (0.006) 0.003 (0.001) PNU-282987 0.35 (0.07)0.16 (0.04) 0.22 (0.04) 0.18 (0.04) 0.066 (0.010)

These results show that Y115F and/or Q139G mutations in the α7 nAChR LBDreduced binding of the endogenous LGIC ligand Ach to the modified LGIC.

Example 5: Mutations that Reduce Associations with Endogenous ReceptorSubunits

Assembly of α7 nAChRs is based on associations of five homomericsubunits through interactions between the LBDs (Celie et al 2004 Neuron41: 907-14). To minimize undesired associations with endogenous α7 nAChRsubunits and/or unwanted associations of chimeric channels, potentialinter-subunit salt bridges were identified by examining the crystalstructure of the acetylcholine binding protein and identifying nearbyinter-subunit residues with opposite charge that also have homologousionic amino acids in the α7 nAChR receptor LBD. Charge reversalmutations (switching the acidic member of a potential salt bridge to abasic residue and its basic partner to an acidic residue) were designedto disrupt inter-subunit interactions with unmodified subunits butpreserve interactions between the subunits with charge reversalmutations (FIG. 6A). Chimeric LGIC subunits having charge reversalmutations were able to assemble selectively with each other withoutinteracting with unmodified channels, e.g. endogenous α7 nAChR. Thedouble mutation of R27D,E41R in the α7 nAChR LBD resulted in functionalchannels (FIG. 6B). Co-expression of these charge reversal channels withα7-5HT3 channels having an unmodified sequence showed that the chargereversal subunits did not co-immunoprecipitate with unmodified channels(FIG. 6C). Combination with potency enhancing mutations andacetylcholine blocking mutations to give the chimeric channelα7^(R27D,E41R,Q79G,Y115F)-GlyR^(A298G) revealed that some agonistsretained high potency for their cognate agonist (Table 4, right column).

These results show that R27D and E41R mutations in α7 nAChR LBD reducedassociation of the modified LGIC subunits with other modified and/orendogenous LGIC subunits.

Example 6: LBD Mutations that Increase Ligand Potency

Mutations in Gly¹⁷⁵ and Pro²¹⁶ of the α7 nAChR LBD in α7-GlyR chimericchannels were tested. Mutation of Gly¹⁷⁵ to Lys (α7G1^(75K)-GlyR) showedincreased potency for ACh (5-fold) (Table 5). For α7^(G175K)-GlyR, itwas also found that nicotine potency was enhanced 10-fold relative tothe unmodified α7-GlyR chimeric channel (Table 5). Mutation of Pro²¹⁶ toIle (α7^(P216I)-GlyR) did not substantially alter ACh potency (Table 5).However, α7^(P216I)-GlyR showed increased nicotine potency by >4-foldrelative to unmodified α7-GlyR (Table 5). These potency enhancingmutations in α7^(G175K)-GlyR and α7^(P216I)-GlyR also affected potencyof several other α7-GlyR agonists up to 30-fold (Table 5). Forα7^(G175K)-GlyR, greater than 10-fold potency enhancement over α7-GlyRwas seen for the clinically used drugs tropisetron, varenicline,cytisine, granisetron, and epibatidine. For α7^(P216I)-GlyR, potencyenhancement was approximately 3-fold (Table 5).

TABLE 5 Agonist potency enhancement by G175K and P216I mutations ata7GlyR chimeric channels. α7GlyR α7GlyR α7GlyR α7GlyR α7GlyR α7GlyRY115F G175K W77F Q79G Compound a7GlyR G175K P216I G175K Y210F G175KG175K Acetylcholine 6.4 (1.2)  1.2 (0.41) 4.0 (0.5)  52 (6.6)  93 (1.3)6.8 (1.6) 4.5 (1.3) Nicotine 5.0 (1.8)  0.5 (0.25) 1.4 (0.1) 4.1 (1.4)  6 (0.5) 1.3 (0.4) 1.1 (0.1) Epibatidine 0.062 (0.021) 0.005 (0.001)0.03 (0.01) 0.036 (0.006) 0.65 (0.11) 0.04 (0)   0.037 (0.013)Varenicline 0.62 (0.2)  0.056 (0.014) 0.18 (0.06) 5.0 (1.7) 4.3 (0.6)0.57 (0.18) 0.42 (0.1)  Cytisine 6.4 (2.0)  0.4 (0.05) 1.9 (0.2) 7.1(1.2) >10 1.5 (0.6) 2.5 (1.1) PNU-282987  0.13 (0.038) 0.005 (0.001) 0.04 (0.004)  0.1 (0.01) 0.7 (0.3) 0.67 (0.35) 0.06 (0.05) Tropisetron 0.15 (0.045) 0.011 (0.002)  0.05 (0.003) 0.027 (0.004) 1.1 (0.2) 0.04(0.01)  0.01 (0.001) Nortropisetron 0.022 (0.007) 0.003 (0.002)  0.006(0.0004) 0.007 (0.001) 0.28 (0.09) 0.004 (0.001) 0.0008 (0.0001)PHA-543613 0.03 (0.01)  0.001 (0.0001) 0.009 (0.001)  0.02 (0.007) 0.26(0.08) 0.041 (0.016)  0.003 (0.0004) Granisetron >100 3.3 (0.1) 6.1(0.9) 1.6 (0.6) 1.4 (0.1) 0.18 (0.02) >100 Ivermectin nd nd nd nd nd ndα7GlyR α7GlyR α7GlyR α7GlyR W77F α7GlyR α7GlyR Q79G Q79G α7GlyR α7GlyRW77F Q79G W77F Q79G Y115F Y115F Y115F Q79G Q79G Y115F G175K G175K G175KG175K G175K Q139L Compound G175K G175K Y210F Y115F Y210F K322L L141FG175K Acetylcholine 41 (3.1) 143 (13)  80 (31) 98 (10) >1000 >200 58 53Nicotine 2.6 (0.7) 6.1 (2.0) 4.2  13 (0.2) >100 14.5 3 5.8 Epibatidine2.6 (2.3) 0.33 0.38  0.22 (0.015) >10 0.27 0.144 0.144 Varenicline 3.3(1.0) >10 >9 >10 >30 >30 >8.1 0.96 Cytisine 6.9 (1.2) 4.025.1 >10 >30 >30 4.74 3.24 PNU-282987 0.5 (0.2) >1 >40 0.08 (0.01) >10.018 0.51 0.05 Tropisetron 0.024 (0.004)  0.1 (0.04) >1 0.027 (0.002)0.717 0.066 0.117 0.105 Nortropisetron 0.0026 (0.0004) 0.014 >12 0.012(0.001) >0.3 0.069 0.075 0.001 PHA-543613 0.12 (0.04) >0.3 >3 0.036(0.006) >1 0.111 0.057 0.024 Granisetron 1.6 (0.4) 0.2 0.06 (0.01) 6.8(1.7) 4.8 >30 0.84 >30 Ivermectin nd nd nd 0.21 nd nd nd nd nd = notdetermined

For use in organisms that produce ACh, it is important to reduce theendogenous ACh potency at these channels comprised of the α7 nAChR LBD.Mutation G175K could be further combined with other mutations thatreduced sensitivity to ACh, such as Y115F and Y210F. Forα7^(Y115F,G175K)-GlyR, high potency for agonists based on tropane orquinuclidine core structures were found for tropisetron, granisetron,nortropisetron, PNU-282987, and PHA-543613, and greatly reduced potencyfor varenicline and cytisine (Table 5). For α7^(G175K,Y210F)-GlyR,potency for most agonists was considerably reduced, however potencyenhancement for granisetron was observed (Table 5).

To develop channels with reduced ACh responsiveness but high potency forother agonists, α7^(G175K)-GlyR was combined with additional mutationsthat increase the potency of specific agonists. Combination with W77Freduced ACh potency, and α7^(W77F,G175K)-GlyR showed increased potencyover α7-GlyR for granisetron, nortropisetron, and tropisetron but notfor PNU282-987, varenicline, cytisine, or PHA-543613 (Table 5).Combination of G175K with Q79G reduced ACh potency, andα7^(Q79G,G175K)-GlyR showed increased potency for nortropisetron,PHA-543613, and tropisetron (Table 5). However, this potency enhancementwas not observed for other agonists, such as PNU282-987, or varenicline.α7^(G175K,Q139L)-GlyR reduced ACh potency and increased potency fornortropisetron and tropisetron (Table 5).

Further reductions in ACh potency were achieved while maintaining highpotency for with synthetic agonists, including those based on tropaneand quinuclidine core structures, by incorporating mutations at W77F,Q79G, L141F, Y115F, G175K, and Y210F in various combinations.α7^(Q79G,Y15F,G175K)-GlyR reduced ACh responsiveness while maintainingpotent responses to tropisetron (Table 5). These mutations also enhancedresponsiveness to other tropane and quinuclidine core structuresrelative to α7^(Y115F,G175K)-GlyR as well as relative to α7-5HT3(representative of endogenous α7 nAChR activity), especiallyquinuclidine thioureas 702 and 703 as well as tropane ester 723, 725,726, 736, 737, 738, and 745 (Table 6). α7^(Q79G,Y115F,G175K)-GlyR alsoshowed high sensitivity to ivermectin (Table 5).α7^(W77F,Q79G,G175K)-GlyR reduced ACh responsiveness while maintaininghigh potency responses to tropisetron, and nortropisetron (Table 5).α7^(W77F,Q79G,G175K)-GlyR also showed enhanced potency for additionaltropane-based core structures, such as compounds 723 and 725, as well asthe clinically used drugs mequitazine and promazine (Table 6).α7^(W77F,G175K,Y210F)-GlyR reduced ACh responsiveness but markedlyimproved potency to granisetron (Table 5). α7^(L141F,Y115F,G175K)-GlyRreduced ACh responsiveness while conferring sensitivity to granisetron(Table 5). α7^(Q79G,Q139L,G175K)-GlyR reduced ACh responsiveness butshowed potent responses to nortropisetron (Table 5).

TABLE 6 Potency enhancement of tropane, quniuclidine agonists,9-azabicyclo[3.3.1]nonane agonists, diazabicyclo[3.2.2]nonane agonists,and promazine by G175K and P216I α7GlyR chimeric channels. Indole andindazole aromatic (A) substituents attached at 3-position. Aromatic C—Xsubstitution Agonist class X₁ X₂ X₃ Y C₁ n C₂ n C₃ n configuration R (A)Quinuclidine N CH₂ NH S 0 1 0 R H 3,5-dichloro- aniline Quinuclidine NCH₂ NH S 0 1 0 R H 3,4-dichloro- aniline Quinuclidine N CH₂ NH S 0 1 0 RH 4-(trifluoro- methoxy)aniline Quinuclidine N CH₂ NH S 0 1 0 R H4-fluoroaniline Quinuclidine N CH₂ NH S 0 1 0 R H 3-chloro-anilineQuinuclidine N CH₂ NH S 0 1 0 R H 3-chloro-2- fluoroaniline QuinuclidineN CH₂ NH S 0 1 0 R H 3-chloro-4- fluoroaniline Quinuclidine N CH₂ NH S 01 0 R H 5-chloro-2- fluoroaniline Quinuclidine N CH₂ NH S 0 1 0 R H3-chloro-4- methylaniline Quinuclidine N CH₂ NH S 0 1 0 R H 5-chloro-2-methylaniline Quinuclidine N CH₂ NH S 0 1 0 S H 4-(trifluoro-methoxy)aniline Tropane C NMe NH S 1 0 0 Endo H 1-methyl- 1H-indoleTropane C NMe O O 1 0 0 Endo H 4-methoxy- 1H-indole Tropane C NMe O O 10 0 Endo H 6-methoxy- 1H-indole Tropane C NMe O O 1 0 0 Endo H7-methoxy- 1H-indole Tropane C NMe O O 1 0 0 Endo H 4-methyl- 1H-indoleTropane C NMe O O 1 0 0 Endo H 7-methyl- 1H-indole Tropane C NMe O O 1 00 Endo H 4-chloro- 1H-indole Tropane C NMe O O 1 0 0 Endo H 5-methoxy-1H-indole Tropane C NMe O O 1 0 0 Endo H 5-chloro- 1H-indole Tropane CNMe O O 1 0 0 Endo H 6-chloro- 1H-indole Tropane C NMe O O 1 0 0 Endo H1H-indazole 9-aza- CH NMe NH O 1 0 1 Endo H 1H-indolebicyclo[3.3.1]nonane 9-aza- CH NMe NH O 1 0 1 Endo H 1H-indazolebicyclo[3.3.1]nonane 9-aza- CH NMe NH O 1 0 1 Endo H 7-methoxy-bicyclo[3.3.1]nonane 1H-indazole 9-aza- CH NH O O 1 0 1 Endo H 1H-indolebicyclo[3.3.1]nonane 1,4-diaza- F dibenzo[b,d]thio- bicyclo[3.2.2]nonanephene 5,5- dioxide 1,4-diaza- NO₂ dibenzo[b,d]thio- bicyclo[3.2.2]nonanephene 5,5- dioxide Quinuclidine N CH₂ CH₂ 0 1 0 R H 10H-pheno- thiazineN,N- 10H-pheno- dimethylpropyl thiazine amine α7GlyR Q79G α7Gly G175Kα7GlyR α7GlyR α7GlyR Q79G Y115F W77F α7GlyR Q79G Y115F G175K R27D Q79GAgonist class Compound α7-5HT3 α7-GlyR G175K G175K G175K Y115F E41RG175K Quinuclidine 677 10.6 4.4 0.66 0.86 3.7 0.98 0.58 nd (0.06)(0.004) (0.7) (0.09) (0.14) Quinuclidine 682 >100 0.2 0.12 0.013 0.400.13 0.06 nd (0.1) (0.001) (0.01) (0.01) (0.012) Quinuclidine 684 >1001.6 0.23 0.078 3.0 0.79 0.4 nd (0.02) (0.022) (0.3) (0.04) (0.03)Quinuclidine 699 2.8 3.6 0.26 0.039    2.9 0.52 0.33 nd (0.11) (0.009)(0.09) (0.1) Quinuclidine 700 1.8 1.9 0.081 0.012    1.5 0.21 0.11 nd(0.009) (0.0002) (0.04) (0.02) Quinuclidine 701 >100 nd 0.47 0.086   5.46 1.0 0.58 nd (0.17) (0.014) (0.2) (0.03) Quinuclidine 702 >1000.9 0.12 0.018    1.6 0.17 0.12 nd (0.004) (0.003) (0.03) (0.02)Quinuclidine 703 >100 nd 0.52 0.03   12.7 1.2 1.1 nd (0.08) (0.01)(0.06) (0.5) Quinuclidine 704 0.7 nd 0.062 0.018 0.76 0.24 0.18 nd(0.008) (0.002) (0.01) (0.02) (0.06) Quinuclidine 705 >100 nd 9.60.67 >10 4.8 4.5 nd (0.14) (1.4) (2.7) Quinuclidine 713 >100 nd 2.10.54 >10    23.9 >10 nd (0.2) (0.06) Tropane 622 >100 nd 0.87 1.3 2.50.93 1.0 1.7 (0.2) (0.4) (0.02) (0.2) Tropane 721 0.5 nd 0.027 0.0150.080 0.020 0.016 0.04 (0) (0.003) (0.002) (0.001) (0.001) Tropane 7220.5 nd 0.02 0.015 0.052 0.028 0.016 0.03 (0.001) (0) (0.008) (0.008)(0.001) Tropane 723 12.8 4 0.31 0.02 0.71 0.07 0.024 0.02 (0.02) (0)(0.46) (0.01) (0.003) Tropane 724 1.2 nd 0.036 0.012 0.091 0.02 0.0120.06 (0.003) (0.002) (0.013) (0.006) (0.002) Tropane 725 12.2 8.1 0.0220.069 0.042 0.022 0.024 (0.02) (0.33) (0.005) (0.0001) Tropane 726 4.2nd 0.58 0.016 0.51 0.044 0.018 0.03 (0.24) (0.001) (0.37) (0.006) (0)Tropane 736 0.83 nd 0.2 0.044 0.57 0.078 0.078 0.06 (0.01) (0.002)(0.21) (0.018) (0.024) Tropane 737 1 0.9 0.082 0.013 0.16 0.033 0.0160.101 (0.004) (0.001) (0.03) (0.004) (0.001) Tropane 738 0.4 nd 0.0150.016 0.04 0.025 0.012 0.033 (0) (0.002) (0.014) (0.002) (0.001) Tropane745 1.2 1.3 0.069 0.026 0.26 0.089 0.043 0.05 (0.002) (0.03) (0.024)(0.014) 9-aza- 749 6.6 nd nd nd nd    1.3 nd 1.9 bicyclo[3.3.1]nonane9-aza- 751 1.8 3.4 nd nd nd    3.2 nd 0.7 bicyclo[3.3.1]nonane 9-aza-760 >100 9.8 nd nd nd    3 nd 1.3 bicyclo[3.3.1]nonane 9-aza- 763 1.90.17 nd nd nd    0.3 nd 0.2 bicyclo[3.3.1]nonane 1,4-diaza- 773 0.1350.001 nd nd    0.0003    0.00042 nd 0.0014 bicyclo[3.2.2]nonane1,4-diaza- 774 0.03 0.006 nd nd    0.00078    0.03 nd 0.03bicyclo[3.2.2]nonane Quinuclidine Mequitazine >30 nd nd nd nd  >10 nd0.15 N,N- Promazine >100 nd nd nd nd >100 nd 1.6 dimethylpropyl amine nd= not determined; parentheses: SEM

α7^(G175K)-GlyR and α7^(P216I)-GlyR along with mutations at Q79G, Y115F,and G175K were also compatible with non-association mutations R27D,E41Ras well as the GlyR IPD mutation A298G, which further enhanced ligandpotency for granisetron, epibatidine, varenicline, cytisine, PNU-282987,tropisetron, nortropisetron, and PHA-543613 (Table 7). Combination withnon-association mutations to form α7^(R27D,E41R,Q79G,Y115F,G175K)further improved the potency for 702, 723, 725, and 726, with low AChresponsiveness (Table 6).

TABLE 7 Agonist potency enhancement by G175K and A298G mutations atα7GlyR chimeric channels as well as W298A at α7GABAc (also referred toas GABA_(A)-ρ) channels. α7GlyR α7GlyR α7GlyR Q79G Q79G α7GlyR α7GlyRα7GlyR Q79G α7GlyR A298G α7GABAc G175K R27D Q79G Q79G A298G Q79G Y115FQ79G Y115F E41R W77F G175K G175K A298G K395 L141F R27D, Q79G CompoundA298G A298G Y115F P216I K396A W298A E41R Y115F Acetylcholine 45 0.66 315 90 52  52 (7.7) >500 Nicotine 3.8 0.11 3.3 1.6 16.5 16.2 4.8(0.4) >39.8 Epibatidine 0.37 0.0023 0.011 0.05 0.15 0.42 0.059 (0.03) 0.267 Varenicline 3.66 0.022 2.37 0.18 >30 6.27 4.9 (0.3) >30 Cytisine14.1 0.134 4.6 5.5 >30 13.3 4.8 (0.4) >30 PNU-282987 1.63 0.00036 0.0090.25 0.11 0.12 0.05 (0.03) 0.34 Tropisetron 0.018 0.0006 0.0028 0.0090.021 0.111 0.013 (0.005) >0.096 Nortropisetron 0.0024 0.00013 0.00840.0012 0.0063 0.009 0.003 (0.001) 0.102 PHA-543613 0.0066 0.00018 0.00390.003 0.0408 0.039 0.0054 0.156 Granisetron 1.2 nd nd nd >30 >100 2.4(0.3) >30 nd = not determined; parentheses: SEMAdditional amino acid substitutions at Gly¹⁷⁵ of the α7 nAChR LBD inα7^(Y115F)-GlyR chimeric channels are also enhanced agonist potency.Potency for tropisetron at α7^(Y115F)-GlyR chimeric channels wasenhanced with additional mutations, which include G175A (7.1-fold),G175F (2-fold), G175H (2.3-fold), G175K (5.6-fold), G175M (2.6-fold),G175R (5.8-fold), G175S (9.3-fold), G175V (16.7-fold).

TABLE 8 Agonist potency enhancement by G175 mutations at α7GlyR Y115Fchimeric channels. α7GlyR α7GlyR α7GlyR α7GlyR α7GlyR α7GlyR α7GlyRα7GlyR Y115F Y115F Y115F Y115F Y115F Y115F Y115F Y115F Compound a7GlyRG175K G175A G175F G175H G175M G175R G175S G175V Acetylcholine 6.4 (1.2) 52 (6.6) 24 67 79 71 29.5 31.5 15 Varenicline 0.62 (0.2)  5.0 (1.7) 5.913.6 12.7 14.1 7.6 9.7 4.6 Tropisetron  0.15 (0.045) 0.027 (0.004) 0.0210.074 0.064 0.057 0.024 0.016 0.009 PHA-543613 0.03 (0.01)  0.02 (0.007)0.027 0.173 0.12 0.25 0.11 0.12 0.037 nd = not determined; parentheses:SEM

Mutations for Leu¹³¹ to smaller amino acids were found to reduce thepotency of canonical agonists ACh and nicotine, while markedlyincreasing potency of varenicline, tropisetron and several otheragonists. α7^(L131A)-GlyR and α7^(L131G)-GlyR had reduced AChresponsiveness (6-fold) and enhanced potency for varenicline (8-fold and17-fold, respectively) and tropisetron (2.5-fold and 3.6-fold,respectively) (Table 9). α7^(L131G)-5HT3 HC had reduced AChresponsiveness (5-fold) and enhanced potency for varenicline (16-fold)and tropisetron (2.3-fold) (FIG. 9A and Table 9). α7^(L131G,Q139L)-GlyRand α7^(L131G,Y217F)-GlyR showed similar potency enhancement overα7-GlyR for varenicline (21-fold) but also reduced ACh sensitivity(−11-fold and −13-fold, respectively). α7^(Q79S,L131G)-GlyR furtherimproved potency over α7-GlyR for varenicline (89-fold) and tropisetron(15-fold). α7^(L131G,Q139L,Y217F)-GlyR showed the greatest improvementin potency over α7-GlyR for varenicline (387-fold) and also showedreduced ACh potency (13-fold) (FIG. 9B and Table 9).α7^(L131G,Q139L,Y217F)-GlyR also showed extremely high potency forcompound 770 (0.001 μM), compound 773 (0.00034 μM), and compound 774(0.00013 μM) (FIG. 10). α7^(Q79S,L131G, Q139L)-GlyR also improvedpotency over α7-GlyR for varenicline (31-fold) and tropisetron (3-fold)but reduced ACh potency (9-fold) (FIG. 9B and Table 9). α7^(L131M)-GlyR,α7^(L131Q)-GlyR, and α7^(L131V)-GlyR reduced ACh potency but enhancedpotency to tropisetron, nortropisetron, PHA-543613, and granisetron(Table 9). α7^(L131F)-GlyR was found to substantially reduced AChpotency but did not improve potency for other agonists (Table 8).α7^(L131G)-GABA_(C) substantially reduced ACh potency but did notimprove potency for other agonists (Table 9).α7^(L131G,Q139L,Y217F)-5HT3 HC (Table 9) improved varenicline potency by131-fold over α7-5HT3 (Table 1). α7^(L131G,Q139L,Y217F)-5HT3 HC alsoshowed high potency for compound 770 (0.007 μM), compound 773 (0.002μM), and compound 774 (0.004 μM) (Table 8).

TABLE 9 Agonist potency enhancement by chimeric channels with L131mutations. α7GlyR α7GlyR α7GlyR α7GlyR L131G α7GlyR α7GlyR Q79S α7GlyRα7GlyR α7GlyR L131G L131G Q139L Q79G Q79S L131G L131G Compound a7GlyRL131A L131G Q139L Y217F Y217F L131G L131G Q139L D219A Acetylcholine 6.442 (21) 41 (11) 68 85 83 (20) >500 21 58 210 (1.2) (3.5) Nicotine 5.0(1.8) 8.0 (3.2) 15 (3.5) 26 28 55 (18) >100 8.2 25  36 (0.8) Epibatidine0.062 0.027 0.009  0.012  0.015 0.021 nd 0.007  0.012  0.16 (0.021)(0.004) (0.002) (0.001) Varenicline 0.62 0.082 0.037  0.03 0.030.0016 >10 0.007 0.02 0.78 (0.2) (0.068) (0.026) (0.001) (0.001) (1.1)(0.001) (0.027) Cytisine 6.4 20.6 13.1 12 30 nd  >30 8.1 10 >30 (2.0)(9.4) (0.66) (0.3) (1.8) PNU-282987 0.13 0.055 0.034  0.063 0.054 0.160.096 0.006  0.018 0.41 (0.038) (0.025) (0.008) (0.03) (0.002) (0.04)Tropisetron 0.15 0.06 0.042  0.13  0.087 0.31    0.09 0.01  0.045  0.36(0.045) (0.021) (0.01) (0.05) (0.003) Nortropisetron 0.022 0.006 0.004 0.024 0.018 0.047 0.012 0.004  0.006 0.07 (0.007) (0.003) (0.001)(0.006) (0.002) (0.008) PHA-543613 0.03 0.012 0.008  0.021 0.016 0.0450.066 0.002  0.009 0.038 (0.01) (0.006) (0.002) (0.008) (0.0005) (0.007)Granisetron >100 17.2 6.7 (1.6)  4  4 nd nd 4.2 nd >30 (12.8) (0.8)765 >100 nd nd nd nd 0.031    0.027 0.024 nd nd (0.02) 770 nd nd nd ndnd 0.001 nd nd nd nd (0.0003) 773    0.001 nd 0.00013  0.00004 nd0.00034    0.00004 nd nd nd 774    0.006 nd 0.00004  0.00004 nd 0.00018   0.00004 nd nd nd αGlyR α75HT3 Q79S L131G L131G αGlyR α75HT3 Q139L α7-αGlyR Q139L αGlyR Y115F αGlyR αGlyR αGlyR L131G Y217F GAβA_(c) CompoundL131F Y217F L131M L131M L131N L131Q L131V HC HC L131G Acetylcholine 92(32) 67 (3)  29 (5)  >500 5 (0.5)   58 16 35 39 >500 Nicotine 20 (6.3)41 (8)  15 nd nd   13 3.9 (0.7) 15 20 >500 Epibatidine 0.24 0.022 0.042nd nd    0.027  0.21  0.009 nd (0.05) (0.004) (0.04) Varenicline 2.60.003  0.53 >100    0.069    0.72  0.33  0.04  0.007    0.3 (0.21)Cytisine    10.5 nd  7 nd nd >30 4.3 (0.7) 11 nd >500 PNU-282987 0.200.05  0.021 nd nd    0.048  0.064  0.033  0.015    0.12 (0.01) (0.018)Tropisetron 0.39 0.084 0.024 0.035    0.025    0.048  0.062  0.066  0.04   0.18 (0.2) (0.009) (0.005) (0.013) Nortropisetron 0.027 0.014  0.006nd nd    0.009  0.003  0.009 nd    0.021 (0.002) (0.001) PHA-543613 0.040.015  0.009    0.028    0.02    0.015  0.011  0.012  0.009    0.027(0.001) (0.002) Granisetron >100 nd  4 nd nd    4 5.4 (1.3)  4 nd >500765 nd 0.034 nd nd >10 nd nd nd  0.11 nd (0.013) 770    0.034 0.001 0.03 nd >10  >0.3 nd nd  0.007 nd (0.0001) 773    0.0005 nd  0.00005 nd   0.0004    0.006 nd nd  0.002 nd    0.0013 nd  0.001 nd    0.0006   0.002 nd nd  0.004 nd nd = not determined; parentheses: SEM

Example 7: Chimeric LGICs in Neurons

AAVs or DNA plasmids containing nucleic acids encoding aα7^(Q79G)-GlyR^(A298G) or α7Q79G,Y115F,G175K-GlyR chimeric LGICs weretransduced into mouse cortical neurons. A low concentration oftropisetron (30 nM or 100 nM) was administered to mouse corticalneurons. Neuron activity was silenced by application of lowconcentration of agonist (FIG. 7 and FIG. 8C).

DNA plasmids containing nucleic acids encoding aα7L131G,Q139L,Y217F-GlyR chimeric LGICs were transfected into mousecortical neurons. Low concentration of varenicline (10 nM) wasadministered to mouse cortical neurons. Neuron activity was silenced byapplication of low concentration of agonist (FIG. 9C).

These results show that modified LGIC activity can be controlled inneurons using low concentration of the LGIC ligands tropisetron andvarenicline.

Example 8: Chimeric LGICs in Therapy

Chemogenetic tools offer an attractive strategy for combined drug andgene therapy. This is because cellular function can be modulated in aconsistent manner across different cell types in various indicationsusing the same ion channels and ligands by use of an exogenouslydelivered ion channel that is selectively engaged by administration of adrug. Identification of ion channels that are gated by well tolerated,clinically used drugs are especially attractive for potentiallyextending chemogenetics to human therapeutic use.

For the drug tropisetron, we have found that it activatesα7^(Q79G)-GlyR^(A298G) with an EC50 of 11 nM, which is similar to thereported IC50 of 10 nM tropisetron for its therapeutic target, the 5HT3receptor (Combrink et al 2009 Pharmacological reports: PR 61: 785-97).

OTHER EMBODIMENTS

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of thedisclosure, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-17. (canceled)
 18. A method of treating a disease or disorder in amammal, the method comprising: administering to a nucleic acid encodinga chimeric ligand gated ion channel (LGIC) subunit to said mammal underconditions in which said modified LGIC subunit can assemble into amodified LGIC comprising said modified LGIC subunit in a cell withinsaid mammal, said modified LGIC subunit comprising: (a) a human alpha7nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD)comprising a Cys-loop domain, wherein said Cys-loop domain comprisesamino acids 150-164 as numbered in SEQ ID NO:2, and (b) a human glycinereceptor (GlyR) ion pore domain (IPD); and administering to the mammal aligand, wherein the ligand acts as an agonist of the modified LGIC. 19.The method of claim 18, wherein the disease or disorder is selected fromthe group consisting of, movement disorder, and spasticity. 20.(canceled)
 21. A method of increasing the activity of a cell in amammal, said method comprising: administering a nucleic acid encoding achimeric LCIG subunit to the cell under conditions in which saidmodified LGIC subunit can assemble into a modified LGIC comprising saidmodified LGIC subunit, said modified LGIC subunit comprising: (a) ahuman α7-nAChR LBD comprising a Cys-loop domain, wherein said Cys-loopdomain comprises amino acids 150-164 as numbered in SEQ ID NO:2, and (b)a human GlyR IPD; and administering an exogenous ligand to the mammal,wherein the exogenous ligand acts as an agonist of the modified LGIC.22. (canceled)
 23. (canceled)
 24. The method of claim 21, wherein theactivity is selected from the group consisting of ion transport, passivetransport, excitation, inhibition, and exocytosis.
 25. The method ofclaim 21, wherein the cell is selected from the group consisting of aneuron, a glial cell, a myocyte, a stem cell, an endocrine cell, and animmune cell. 26-35. (canceled)
 36. The method of claim 18, wherein thenucleic acid encoding the chimeric LGIC receptor is delivered to themammal in a viral vector.
 37. The method of claim 36, wherein the viralvector is selected from the group consisting of an adeno-associatedvirus, a herpes simplex virus, and a lentivirus vector.
 38. The methodof claim 18, wherein the nucleic acid encoding the chimeric LGICreceptor is operably linked to a promoter.
 39. The method of claim 38,wherein the promoter is a neuron-specific promoter, a muscle-specificpromoter, or a glial-specific promoter.
 40. The method of claim 18,wherein the nucleic acid encoding the chimeric LGIC receptor isadministered by oral, subcutaneous, intracranial, intravenous, ortopical administration.
 41. The method of claim 18, wherein the mammalis a human.
 42. The method of claim 21, wherein said exogenous ligand isa synthetic ligand.
 43. The method of claim 21, wherein said modifiedLGIC has reduced sensitivity to an endogenous ligand.
 44. The method ofclaim 21, wherein the mammal is a human.
 45. A method of decreasing theactivity of a cell in a mammal, said method comprising: administering anucleic acid encoding a chimeric LCIG subunit to the cell underconditions in which said modified LGIC subunit can assemble into amodified LGIC comprising said modified LGIC subunit, said modified LGICsubunit comprising: (a) a human α7-nAChR LBD comprising a Cys-loopdomain, wherein said Cys-loop domain comprises amino acids 150-164 asnumbered in SEQ ID NO:2, and (b) a human GlyR IPD; and administering anexogenous ligand to the mammal, wherein the exogenous ligand acts as anantagonist of the modified LGIC.
 46. The method of claim 45, wherein theactivity is selected from the group consisting of ion transport, passivetransport, excitation, inhibition, and exocytosis.
 47. The method ofclaim 45, wherein the cell is selected from the group consisting of aneuron, a glial cell, a myocyte, a stem cell, an endocrine cell, and animmune cell.
 48. The method of claim 45, wherein said exogenous ligandis a synthetic ligand.
 49. The method of claim 45, wherein said modifiedLGIC has reduced sensitivity to an endogenous ligand.
 50. The method ofclaim 45, wherein the mammal is a human.